Thermophilic dna polymerase mutants

ABSTRACT

This disclosure relates to thermophilic family B DNA polymerases comprising a neutral amino acid residue at a certain position near the C-terminus of the catalytic domain, which corresponds to a position occupied by a basic amino acid residue in wild-type Pfu polymerase. The thermophilic family B DNA polymerases provided herein also comprise an N-terminal domain comprising a uracil-binding pocket that has been modified to reduce template uracil binding. Related uses, methods, and compositions are also provided. In some embodiments, the polymerases comprise a 3′-5′ exonuclease domain and/or a sequence nonspecific dsDNA binding domain.

RELATED APPLICATIONS

This application is a continuation of U.S. Pat. Application No.16/623332, filed on Dec. 16, 2019, which is a 371 U.S. National PhaseApplication of PCT/EP2018/066896 filed Jun. 25, 2018, which claimspriority to and the benefit of U.S. Provisional Pat. Applications Serial# 62/524,730 filed Jun. 26, 2017, which applications are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and single letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an XML file inthe form of the file named “9748-108598-02_ST_26.xml” (~418,221 bytes),which was created on Feb. 22, 2023 which is incorporated by referenceherein.

BACKGROUND OF THE DISCLOSURE

This disclosure relates to the field of thermophilic DNA polymerasemutants, including methods, uses, and compositions thereof.

Thermophilic DNA polymerases are commonly used in biotechnology andmolecular biology applications, including nucleic acid synthesistechniques such as amplification (e.g., polymerase chain reaction, orPCR), which involves cycles of alternating denaturation and primerannealing and extension. Thermophilic DNA polymerases are resistant toinactivation by high temperatures and so are compatible with thermaldenaturation steps. DNA polymerases comprise a catalytic domain thatextends a 3′ terminus of a DNA strand in a template-dependent manner.DNA polymerases can also comprise an exonuclease domain, such as a 3′ to5′ exonuclease domain. Such an exonuclease domain can reduce thefrequency of misincorporation by removing mismatched nucleotides fromthe 3′ end of a nascent DNA strand. Certain artificial DNA polymerasesfurther comprise a sequence non-specific double-stranded DNA (dsDNA)binding domain. The presence of this domain can improve performance ofthe enzyme with respect to various parameters, including processivity,sensitivity, and yield.

Nucleic acid amplification can permit rapid detection of a targetnucleic acid sequence and/or provide sufficient quantities of a samplefor further analysis or manipulation, such as sequencing, cloning,restriction digestion, hybridization, ligation, mutagenesis,recombination, etc. Two key parameters of amplification are sensitivityand yield. Improving the sensitivity reduces the minimum amount of atarget needed to produce a detectable product. Improving the yieldincreases the amount of product that results from a reaction, or reducesthe amount of time and/or reagents necessary to obtain a given amount ofproduct.

Samples may be refractory to amplification or may decrease sensitivityand/or yield if they contain nucleic acid synthesis inhibitors, whichmay occur naturally in the sample or may be introduced during earliersample processing steps. Examples of nucleic acid synthesis inhibitorsinclude polyanions such as heparin or xylan; anionic detergents such assodium dodecyl sulfate; and certain complex organic substances such ashumic acid, collagen, heme and heme-containing proteins, bile salts, andthe like. Thermophilic DNA polymerases with improved tolerance of suchinhibitors would reduce the need for purification and other sampleprocessing steps in advance of nucleic acid synthesis and reduce thefrequency of unsatisfactory synthesis reactions.

Certain polymerases such as the family B polymerases, includingPyrococcus furiosus (Pfu) DNA polymerase (see Kennedy et al., “TheMechanistic Architecture of the Thermostable Pyrococcus furiosus FamilyB DNA Polymerase Motif A and its Interaction with dNTP Substrate,”Biochemistry 2009 December 1; 48(47): 11161-11168.doi:10.1021/bi9010122) and related polymerases, may benefit frommutations that increase yield and/or sensitivity. In some instances, anA408S mutation has been introduced into family B polymerases in order toimprove accuracy (i.e., reduced error rate or increased fidelity), butwith a detrimental impact on yield and/or sensitivity. It would bedesirable to provide variants of family B polymerases that have improvedyield and/or sensitivity. Further, coupled with an A408S mutation, suchvariants may have improved yield and/or sensitivity and also improvedfidelity. It would also be desirable to provide variants of suchpolymerases with improved inhibitor resistance. Such polymerases couldbe suitable for use with a broader spectrum of samples and/or couldreduce the need for preprocessing in advance of nucleic acid synthesisreactions in which high fidelity is desirable, such as for cloning,sequencing, gene construction, site-directed mutagenesis, etc.

A feature of archaeal family B DNA polymerases is the ability torecognize and bind uracil bases in template DNA during the amplificationreaction. The uracil-binding pocket in nature reduces the accumulationof mutations caused by cytosine deamination to uracil and subsequent G-Cbase pair transitions to A-T during DNA replication. The uracil bindingpocket recognizes and binds uracil bases in the template strand,stopping the polymerase. In PCR, the uracil-binding property of archaealfamily B polymerases may be disadvantageous and result in decreased DNAamplification yields and lowered sensitivity. Even trace amounts ofuracil may decrease DNA amplification yields and lower the sensitivityin simple PCR, high-fidelity PCR, and particularly in long-range PCR,where long elongation times are required. Furthermore, in certaindiagnostic methods, qPCR, RT-qPCR, and end-point PCR may be performedusing dNTP mixtures in which dTTP is partially or fully replaced bydUTP. Uracil-DNA glycosylase treatment and subsequent heating of thesamples is used to degrade the DNA containing uracil and preventcarryover contamination, a primary concern in diagnostic laboratories. Athermostable archaeal family B DNA polymerase with improved yield and/orsensitivity and/or fidelity, and in which termplate uracil binding isdiminished or abolished would therefore be highly desirable.

The uracil-binding pocket is located in the N-terminal domain ofarchaeal family B DNA polymerases and comprises amino acids from twoconserved regions of the archaeal DNA polymerases: Region A and RegionB, which are separated by a less conserved region. In the archaealpolymerase from Pyrococcus furiosus, Region A comprises amino acids 1-40and Region B comprises amino acids 78-130. Uracil binding is mediated byrelatively inflexible main-chain atoms, consistent with the sizeabledifference (greater than 2 orders of magnitude) in binding affinity foruracil- and non-uracil-containing DNA. The pocket also contains arelatively high proportion of prolines, which may impart additionalrigidity. The C5-C6 edge of the bound uracil packs against Pro90, Pro36and Phe116. Packing above and below uracil are the side chains of Val93and Pro36, and Ile114 and Arg119, respectively. These amino acids show ahigh level of conservation, approaching 100%, emphasizing theirstructural and functional importance (Firbank, 2008, J. Mol. Biol. 381:529-539).

Archaeal family B DNA polymerases with substitutions at position Pro36have been shown to have reduced affinity for uracil as compared to thewild-type polymerase. P36A mutant of Pfu DNA polymerase was able toextend primers beyond template strand uracil, yielding a mixture oftruncated and full-length products (Firbank 2008). P36L mutant of Sh1BDNA polymerase was shown to amplify DNA in the presence of higherconcentrations of dUTP compared with the wild-type enzyme, while P36Hshowed the highest resistance by performing PCR in the reaction mixtureswhere dTTP was completely replaced by dUTP (Tubeleviciute, 2010, ProteinEngineering Design & Selection 23: 589-597).

Thus, there are needs for thermophilic DNA polymerases having increasedinhibitor tolerance and/or the capability to provide increased yieldand/or sensitivity and/or fidelity, and in which template uracil bindingis diminished or abolished. Provided herein are polymerases and relatedmethods and compositions that can solve these needs and/or provide otherbenefits.

In some embodiments, the present disclosure provides thermophilic DNApolymerase mutants and methods of nucleic acid synthesis usingthermophilic DNA polymerase mutants. In some embodiments, a thermophilicDNA polymerase comprising a family B polymerase N-terminal domaincomprising a uracil-binding pocket and a family B polymerase catalyticdomain is provided, the family B polymerase N-terminal domain comprisinga uracil-binding pocket having an amino acid sequence in which theposition corresponding to position 36 of SEQ ID NO: 1 is any amino acidother than P, and the family B polymerase catalytic domain having anamino acid sequence in which the position corresponding to position 762of SEQ ID NO: 1 is a neutral amino acid residue. In some embodiments,the position corresponding to position 36 of SEQ ID NO: 1 is selectedfrom Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, and G. In someembodiments, the position corresponding to position 36 of SEQ ID NO: 1is H. In some embodiments, the position corresponding to position 762 ofSEQ ID NO: 1 is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V,P, and G. In some embodiments, the position corresponding to position762 of SEQ ID NO: 1 is selected from Q and N.

In some embodiments, the family B polymerase catalytic domain has atleast 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the family Bpolymerase catalytic domain sequence of a sequence selected from SEQ IDNOs: 6 to 10, 15 to 18, 25, 26, 33, 34, 37, 38, 41, 42, and 45 to 48,wherein X is the neutral amino acid residue and is selected from Q, N,H, S, T, Y, C, M, W, A, I, L, F, V, P, and G. In some embodiments, X isN or G.

In some embodiments, a thermophilic DNA polymerase is provided, whereinthe family B polymerase N-terminal domain comprising a uracil-bindingpocket has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity tothe family B polymerase N-terminal domain comprising a uracil-bindingpocket sequence of a sequence selected from SEQ ID NOs: 115 to 121 and162 to 168, wherein X¹ is any amino acid other than P. In someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, and G. In some embodiments, X¹ is H.

In some embodiments, a thermophilic DNA polymerase is provided,comprising a family B polymerase N-terminal domain comprising auracil-binding pocket and a family B polymerase catalytic domain,wherein the amino acid residue at the position of the amino acidsequence that aligns to position 36 of SEQ ID NO: 1 is any amino acidother than P, and wherein the amino acid residue at the position of theamino acid sequence that aligns to position 762 of SEQ ID NO: 1 is aneutral amino acid residue.. In some embodiments, the amino acid residueat the position of the amino acid sequence that aligns to position 36 ofSEQ ID NO: 1 is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V,and G. In some embodiments, the amino acid residue at the position ofthe amino acid sequence that aligns to position 36 of SEQ ID NO: 1 is H.In some embodiments, the amino acid residue at the position of the aminoacid sequence that aligns to position 762 of SEQ ID NO: 1 is selectedfrom Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G. in someembodiments, the amino acid residue at the position of the amino acidsequence that aligns to position 762 of SEQ ID NO: 1 is selected from Qand N.

In some embodiments, the family B polymerase catalytic domain has atleast 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 6. Insome embodiments, the family B polymerase N-terminal domain comprising auracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to a sequence selected from SEQ ID NOs: 115 to 121 and 162 to168, wherein X¹ is any amino acid other than P. In some embodiments, X¹is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, and G. Insome embodiments, X¹ is H.

In some embodiments, the amino acid residue at the position of the aminoacid sequence that corresponds to position 408 of SEQ ID NO: 1 is aserine.

In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket comprises a consecutive amino acidsequence of RHYIY (SEQ ID NO: 177), QHYIY (SEQ ID NO: 178), EHYIY (SEQID NO: 179), EHYFY (SEQ ID NO: 180), or RHYFY (SEQ ID NO: 181), and thefamily B polymerase catalytic domain comprises a consecutive amino acidsequence of WQKTX (SEQ ID NO: 182), XQTGL (SEQ ID NO: 183), KTXQT (SEQID NO: 184), YQKTX (SEQ ID NO: 185), XQVGL (SEQ ID NO: 186), KTXQV (SEQID NO: 187), YQSSX (SEQ ID NO: 188), XQTGL (SEQ ID NO: 183), SSXQT (SEQID NO: 189), TGRVX (SEQ ID NO: 190), XKSLL (SEQ ID NO: 191), RVXKS (SEQID NO: 192), TGRSX (SEQ ID NO: 193), XRTLL (SEQ ID NO: 194), or RSXRT(SEQ ID NO: 195);

wherein X is a neutral amino acid residue; and wherein X is within 20residues of the C-terminus of the family B polymerase catalytic domain.In some embodiments, the family B polymerase catalytic domain is asubfamily B3 polymerase domain. In some embodiments, the neutral aminoacid residue is a polar neutral amino acid residue. In some embodiments,the neutral amino acid residue comprises an amide. In some embodiments,the neutral amino acid residue is selected from Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, and G. In some embodiments, the neutral amino acidresidue is selected from Q and N. In some embodiments, the neutral aminoacid residue is Q.

In some embodiments, the thermophilic DNA polymerase comprises asequence non-specific double-stranded DNA-binding domain. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to a sequence selected from SEQ IDNOs: 53 to 62. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain is C-terminal to the family Bpolymerase catalytic domain. In some embodiments, the sequencenon-specific double-stranded DNA-binding domain is a 7 kD DNA-bindingdomain. In some embodiments, the sequence non-specific double-strandedDNA-binding domain is an Sso7d, Sac7d, or Sac7e domain.

In some embodiments, the thermophilic DNA polymerase comprises: (a) theconsecutive amino acid residues LDFRS (SEQ ID NO: 196), (b) theconsecutive amino acid residues FRSLY (SEQ ID NO: 197), or (c) theconsecutive amino acid residues SLYPS (SEQ ID NO: 198), wherein theunderlined serine residue is within 30 amino acid residues of theN-terminus of the family B polymerase catalytic domain.

In some embodiments, the thermophilic DNA polymerase comprises a 3′ to5′ exonuclease domain. In some embodiments, the 3′ to 5′ exonucleasedomain is N-terminal to the family B polymerase catalytic domain. Insome embodiments, the 3′ to 5′ exonuclease domain is a DEDDy archaealexonuclease domain. In some embodiments, the 3′ to 5′ exonuclease domaincomprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or100% identity to SEQ ID NO: 63. In some embodiments, the 3′ to 5′exonuclease domain comprises an amino acid sequence having at least 90%,95%, 98%, 99%, or 100% identity to the 3′ to 5′ exonuclease domain of asequence selected from SEQ ID NOs: 1, 19, 23, 31, 35, 39, 43, 49, 51,52, 76 to 79, 92, 96, 102, 104, 106, 108, 110, 112, and 113, 139, 143,149, 151, 155, 157, 159, and 160.

In some embodiments, the thermophilic DNA polymerase comprises an aminoacid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to asequence selected from SEQ ID NOs: 80 to 113 and 127 to 160, wherein X¹is any amino acid other than P and X² is the neutral amino acid residue.In some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A,I, L, F, V, G. In some embodiments, X¹ is H. In some embodiments, X² isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G. Insome embodiments, X² is N or G.

In some embodiments, the thermophilic DNA polymerase comprises an aminoacid sequence comprising (i) at least one difference at a positioncorresponding to position 15, 72, 93, 141, 143, 247, 265, 337, 385, 387,388, 399, 400, 405, 407, 410, 485, 542, 546, 593, or 595 of SEQ ID NO: 1or (ii) at least one missing residue corresponding to position 92, 93,94, or 381 of SEQ ID NO: 1. In some embodiments, the at least onemismatch or missing residue comprises at least one of:

-   (i) a missing residue corresponding to position 92 or 94 of SEQ ID    NO: 1;-   (ii) a Q or R at the position corresponding to position 93 of SEQ ID    NO: 1;-   (iii) an A at the position corresponding to position 141 of SEQ ID    NO: 1;-   (iv) an A at the position corresponding to position 143 of SEQ ID    NO: 1;-   (v) an I at the position corresponding to position 337 of SEQ ID NO:    1;-   (vi) a Q, S, N, L, or H at the position corresponding to position    385 of SEQ ID NO: 1;-   (vii) a P or S at the position corresponding to position 387 of SEQ    ID NO: 1;-   (viii) a P at the position corresponding to position 388 of SEQ ID    NO: 1;-   (ix) a D at the position corresponding to position 399 of SEQ ID NO:    1;-   (x) a G or D at the position corresponding to position 400 of SEQ ID    NO: 1;-   (xi) an E at the position corresponding to position 405 of SEQ ID    NO: 1;-   (xii) an I at the position corresponding to position 407 of SEQ ID    NO: 1;-   (xiii) an L or F at the position corresponding to position 410 of    SEQ ID NO: 1;-   (xiv) a T at the position corresponding to position 485 of SEQ ID    NO: 1;-   (xv) a P at the position corresponding to position 542 of SEQ ID NO:    1;-   (xvi) an H at the position corresponding to position 546 of SEQ ID    NO: 1;-   (xvii) a T at the position corresponding to position 593 of SEQ ID    NO: 1; or-   (xviii) an S at the position corresponding to position 595 of SEQ ID    NO: 1.

In some embodiments, the thermophilic DNA polymerase has at least one ofthe following properties:

-   (i) capable of amplifying a 2 kb target from 40 ng of human genomic    DNA template in the presence of 0.2 µM heparin in a PCR; and/or-   (ii) capable of amplifying a 2 kb target from 40 ng of human genomic    DNA template in the presence of 400 ng/µl xylan in a PCR;

wherein amplification is successful if product is detectable by agarosegel electrophoresis and ethidium bromide staining within 30 PCR cycles.

In some embodiments, the thermophilic DNA polymerase is capable ofamplifying a 2 kb target from 200 ng of human genomic DNA in thepresence of at least 100 µM, at least 120 µM, at least 140 µM, at least160 µM, at least 180 µM, or at least 200 µM dUTP, wherein amplificationis successful if product is detectable by agarose gel electrophoresisand ethidium bromide staining within 30 PCR cycles.

In some embodiments, the thermophilic DNA polymerase is bound to athermolabile inhibitor. In some embodiments, the thermolabile inhibitorcomprises an antibody, an Affibody®, an oligonucleotide, such as anaptamer, and/or a chemical modification.

In some embodiments, a method of in vitro nucleic acid synthesis isprovided, comprising contacting at least one primer and at least onetemplate with a thermophilic DNA polymerase provided herein in thepresence of at least one dNTP. In some embodiments, the thermophilic DNApolymerase is initially bound to a thermolabile inhibitor and the methodcomprises denaturing the inhibitor. In some embodiments, the methodfurther comprises amplification of the template. In some embodiments,the amplification comprises a PCR.

In some embodiments, a nucleic acid comprising a sequence encoding athermophilic DNA polymerase described herein is provided. In someembodiments, an expression vector comprising the nucleic acid isprovided. In some embodiments, an isolated host cell comprising thenucleic acid or the expression vector is provided. In some embodiments,a method of producing a thermophilic DNA polymerase described herein isprovided, comprising culturing at least one host cell comprising anucleic acid encoding the thermophilic DNA polymerase, wherein the atleast one host cell expresses the thermophilic DNA polymerase. In someembodiments, the method further comprises isolating the thermophilic DNApolymerase.

In some embodiments, compositions comprising thermophilic DNApolymerases described herein are provided. In some embodiments, thecomposition comprises at least one hot start inhibitor. In someembodiments, the composition comprises at least two hot startinhibitors. In some embodiments, each hot start inhibitor isindependently selected from an antibody, an Affibody®, anoligonucleotide and/or a chemical modification. In some embodiments, thecomposition comprises at least two antibodies. In some embodiments, thecomposition comprises an antibody and an oligonucleotide. In someembodiments, the oligonucleotide is an aptamer. In some embodiments, thecomposition comprises at least one antibody, and an Affibody® or anaptamer.

In some embodiments, the composition is a storage composition. In someembodiments, the composition comprises at least one protein stabilizer.In some embodiments, the protein stabilizer is selected from BSA,inactive polymerase, and apotransferrin. In some embodiments, thecomposition comprises a UTPase. In some embodiments, the compositioncomprises at least one buffering agent. In some embodiments, thebuffering agent is selected from acetate buffer, sulfate buffer,phosphate buffer, MOPS, HEPES and Tris-(hydroxymethyl)aminomethane(TRIS). In some embodiments, the composition comprises at least onemonovalent cationic salt. In some embodiments, the monovalent cationicsalt is selected from KCl and NaCl. In some embodiments, the compositioncomprises at least one stabilizer. In some embodiments, the stabilizeris selected from glycerol, trehalose, lactose, maltose, galactose,glucose, sucrose, dimethyl sulfoxide (DMSO), polyethylene glycol, andsorbitol. In some embodiments, the composition comprises at least onereducing agent. In some embodiments, the reducing agent isdithiothreitol (DTT). In some embodiments, the composition comprises atleast one divalent chelating agent. In some embodiments, the divalentchelating agent is EDTA. In some embodiments, the composition comprisesat least one detergent. In some embodiments, the detergent is anionic.In some embodiments, the detergent is cationic. In some embodiments, thedetergent is non-ionic. In some embodiments, the detergent iszwitterionic. In some embodiments, the composition comprises a detergentselected from Hecameg(6-O-(N-Heptylcarbamoyl)-methyl-α-D-glucopyranoside), Triton X-200,Brij-58, CHAPS, n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate(SDS), TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON®X-102, TRITON® X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405,TRITON® X-705, Tween® 20 and/or ZWITTERGENT®.

In some embodiments, the composition is an aqueous solution. In someembodiments, the composition is a lyophilized composition.

In some embodiments, the composition is a reaction composition. In someembodiments, the composition comprises at least one buffering agent. Insome embodiments, the buffering agent is selected from acetate buffer,sulfate buffer, phosphate buffer, MOPS, HEPES andTris-(hydroxymethyl)aminomethane (TRIS). In some embodiments, thecomposition comprises at least one monovalent cationic salt. In someembodiments, the monovalent cationic salt is selected from KCl and NaCl.In some embodiments, the composition comprises at least one divalentcationic salt. In some embodiments, the divalent cationic salt is MgCl₂or MnCl₂. In some embodiments, the composition comprises at least onedetergent. In some embodiments, the detergent is anionic. In someembodiments, the detergent is cationic. In some embodiments, thedetergent is non-ionic. In some embodiments, the detergent iszwitterionic. In some embodiments, the composition comprises a detergentselected from Hecameg(6-O-(N-Heptylcarbamoyl)-methyl-α-D-glucopyranoside), Triton X-200,Brij-58, CHAPS, n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate(SDS), TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON®X-102, TRITON® X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405,TRITON® X-705, Tween® 20 and/or ZWITTERGENT®. In some embodiments, thecomposition comprises at least one dNTP. In some embodiments, thecomposition comprises dATP, dGTP, dTTP, and dCTP. In some embodiments,the composition further comprises glycerol, DMSO, and/or ammoniumsulphate. In some embodiments, the composition comprises at least onedye. In some embodiments, the composition comprises at least one dyeselected from xylene cyanol FF, tartrazine, phenol red, quinolineyellow, zylene cyanol, Brilliant Blue, Patent Blue, indigocarmine, acidred 1, m-cresol purple, cresol red, neutral red, bromocresol green, acidviolet 5, bromo phenol blue, and orange G. In some embodiments, thecomposition comprises at least one agent that increases the density ofthe composition. In some embodiments, the composition comprises at leastone agent selected from PEG 4000 and/or sucrose. In some embodiments,the composition comprises at least one primer. In some embodiments, thecomposition comprises at least one nucleic acid template.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of PCR amplifications in which heparin waspresent at a series of concentrations from 0 to 0.3 µM and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain with (“762Q”) or without (“762K”) a neutral amino acid residue atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) and with(“408S”) or without (no 408 designation) a serine at the position thataligns to position 762 of Pfu (SEQ ID NO: 1; aligning to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 2 shows a comparison of PCR amplifications in which xylan waspresent at a series of concentrations from 0 to 400 ng/µl and in whichthe polymerase comprised a family B thermophilic DNA polymerasecatalytic domain with (“762Q”) or without (“762K”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with (“408S”) or without (no 408 designation) a serine at theposition that aligns to position 762 of Pfu (SEQ ID NO: 1; aligning toposition 25 of the Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 3 shows a comparison of PCR amplifications in which humic acid waspresent at a series of concentrations from 0 to 1 ng/µl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain with (“762Q”) or without (“762K”) a neutral amino acid residue atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 379 of the Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 4 shows a comparison of PCR amplifications in which sodium dodecylsulfate (“SDS”) was present at a series of concentrations from 0 to0.016% or 0.2% (w/v) and in which the polymerase comprised a family Bthermophilic DNA polymerase catalytic domain with (“762Q”) or without(“762K”) a neutral amino acid residue at the position that aligns toposition 762 of Pfu (SEQ ID NO: 1; aligning to position 379 of the Pfucatalytic domain (SEQ ID NO: 6)).

FIG. 5 shows a comparison of PCR amplifications in which a 2 kb fragmentwas amplified from a series of amounts of human genomic DNA templatebetween 0 and 400 ng in a 20 µl PCR mixture using a polymerasecomprising a family B thermophilic DNA polymerase catalytic domain with(“762Q”) or without (“762K”) a neutral amino acid residue at theposition that aligns to position 762 of Pfu (SEQ ID NO: 1; aligning toposition 379 of the Pfu catalytic domain (SEQ ID NO: 6)) and with(“408S”) or without (no 408 designation) a serine at the position thataligns to position 762 of Pfu (SEQ ID NO: 1; aligning to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 6A shows a comparison of PCR amplifications in which a 10 kbfragment was amplified from a series of amounts of bacteriophage lambdaDNA template between 0 and 200 ng in a 20 µl PCR mixture using apolymerase comprising a family B thermophilic DNA polymerase catalyticdomain with (“762Q”) or without (“762K”) a neutral amino acid residue atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) and with(“408S”) or without (no 408 designation) a serine at the position thataligns to position 762 of Pfu (SEQ ID NO: 1; aligning to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 6B shows a bar graph illustrating yield from amplification of a 10kb fragment from a series of amounts of bacteriophage lambda DNAtemplate using a polymerase comprising a family B thermophilic DNApolymerase catalytic domain with (“762Q”) or without (“762K”) a neutralamino acid residue at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 379 of the Pfu catalytic domain (SEQID NO: 6)) and with (“408S”) or without (no 408 designation) a serine atthe position that aligns to position 762 of Pfu (SEQ ID NO: 1; aligningto position 25 of the Pfu catalytic domain (SEQ ID NO: 6)).

FIG. 7A shows a comparison of PCR amplifications of a 2 kb product inwhich human genomic DNA template was present at a series of amounts from0 to 400 ng in a reaction volume of 20 µl and in which the polymerasecomprised a family B thermophilic DNA polymerase catalytic domainwithout (“408S 762K”) or with (“408S 762Q”) a neutral amino acid residueat the position that aligns to position 762 of Pfu (SEQ ID NO: 1;aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) andwith a serine at the position that aligns to position 762 of Pfu (SEQ IDNO: 1; aligning to position 25 of the Pfu catalytic domain (SEQ ID NO:6)).

FIG. 7B shows a comparison of PCR amplifications of a 5 kb product inwhich bacteriophage lambda DNA template was present at a series ofamounts from 0 to 200 ng in a reaction volume of 20 µl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain without (“408S 762K”) or with (“408S 762Q”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with a serine at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 25 of the Pfu catalytic domain (SEQID NO: 6)).

FIG. 8 shows a comparison of PCR amplifications of a 20 kb product inwhich bacteriophage lambda DNA template was present at a series ofamounts from 0 to 100 ng in a reaction volume of 20 µl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain without (“408S 762K”) or with (“408S 762Q”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with a serine at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 25 of the Pfu catalytic domain (SEQID NO: 6)).

FIG. 9 shows a comparison of PCR amplifications of a 20 kb product inwhich Escherichia coli genomic DNA template was present at a series ofamounts from 0 to 40 ng in a reaction volume of 20 µl and in which thepolymerase comprised a family B thermophilic DNA polymerase catalyticdomain without (“408S 762K”) or with (“408S 762Q”) a neutral amino acidresidue at the position that aligns to position 762 of Pfu (SEQ ID NO:1; aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6))and with a serine at the position that aligns to position 762 of Pfu(SEQ ID NO: 1; aligning to position 25 of the Pfu catalytic domain (SEQID NO: 6)).

FIG. 10 shows a comparison of PCR amplifications of a 7.5 kb product inwhich human genomic DNA template was present at a series of amounts from0 to 400 ng in a reaction volume of 20 µl and in which the polymerasecomprised a family B thermophilic DNA polymerase catalytic domainwithout (“408S 762K”) or with (“408S 762Q”) a neutral amino acid residueat the position that aligns to position 762 of Pfu (SEQ ID NO: 1;aligning to position 379 of the Pfu catalytic domain (SEQ ID NO: 6)) andwith a serine at the position that aligns to position 762 of Pfu (SEQ IDNO: 1; aligning to position 25 of the Pfu catalytic domain (SEQ ID NO:6)).

FIGS. 11A through 11B show a multiple amino acid sequence alignment ofThermococcus litoralis (“Tli” ; SEQ ID NO: 31), (“Tsp9N7”; SEQ ID NO:49), Thermococcus gorgonarius (“Tgo”; SEQ ID NO: 39), Thermococcuskodakarensis (“Tko” ; SEQ ID NO: 43), Pyrococcus furiosus (“Pfu” ; SEQID NO: 2), and Deep Vent (“DP”; SEQ ID NO: 23) polymerases, in which theposition corresponding to position 36 of Pfu (SEQ ID NO: 1) is markedwith a percent (%), the position corresponding to position 408 of Pfu(SEQ ID NO: 1) is marked with an asterisk (*) and the positioncorresponding to position 762 of Pfu (SEQ ID NO: 1) is marked with apound (#). The position corresponding to position 762 of Pfu isindicated as “X” in each amino acid sequence of the sequence alignment.X may be selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, andG; in some embodiments, X is selected from Q and N. In some embodiments,X is Q.

FIG. 12 shows a multiple amino acid sequence alignment of the catalyticdomains of Thermococcus litoralis (“Tli” ; SEQ ID NO: 33), (“Tsp9N7”;SEQ ID NO: 47), Thermococcus gorgonarius (“Tgo”; SEQ ID NO: 41),Thermococcus kodakarensis (“Tko” ; SEQ ID NO: 45), Pyrococcus furiosus(“Pfu” ; SEQ ID NO: 7), and Deep Vent (“DP”; SEQ ID NO: 25) polymerases,in which the position corresponding to position 408 of Pfu in thefull-length polymerase (SEQ ID NO: 1; corresponding to position 25 ofthe Pfu catalytic domain (SEQ ID NO: 6)) is marked with an asterisk (*)and position corresponding to position 762 of Pfu in the full-lengthpolymerase (SEQ ID NO: 1; corresponding to position 379 in the Pfucatalytic domain (SEQ ID NO: 6)) is marked with a pound (#). Theposition corresponding to position 762 of Pfu (SEQ ID NO: 1; position379 in the Pfu catalytic domain (SEQ ID NO: 6)) is indicated as “X” inthe sequence alignment. X may be selected from Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, and G; in some embodiments, X is selected from Qand N. In some embodiments, X is Q.

FIG. 13 shows amplification of a 2 kb human genomic DNA product by a 36H408S 762Q polymerase in the presence increasing replacement of dTTP withdUTP.

FIG. 14 shows amplification of a 5 kb human genomic DNA product by a 36H408S 762Q polymerase in the presence increasing replacement of dTTP withdUTP.

FIG. 15 shows amplification of a 2 kb human genomic DNA product by a 36H408S 762Q polymerase and 36H polymerase in the presence of added dUTP

FIG. 16 shows multiplex amplification of human genomic DNA with 4 or 5primer pairs.

DETAILED DESCRIPTION

This description and exemplary embodiments should not be taken aslimiting. For the purposes of this specification and appended claims,unless otherwise indicated, all numbers expressing quantities,percentages, or proportions, and other numerical values used in thespecification and claims, are to be understood as being modified in allinstances by the term “about,” to the extent they are not already somodified. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the following specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” and any singular use of anyword, include plural referents unless expressly and unequivocallylimited to one referent. As used herein, the term “include” and itsgrammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

The term “nucleic acid synthesis” refers to template-directed synthesisof a nucleic acid strand using a polymerase enzyme. Nucleic acidsynthesis includes all such template-directed nucleic acid synthesis bya polymerase, including, but not limited to, amplification, PCR, endpoint PCR (epPCR), real time or quantitative PCR (qPCR), one-stepRT-PCR, sequencing, etc.

As used herein the terms “amplify”, “amplifying”, “amplification” andother related terms include producing multiple copies of an originalbiomolecule, such as a nucleic acid. In some embodiments, nucleic acidamplification produces multiple copies of an original nucleic acidand/or its complement (e.g., target nucleic acid, also referred to as atarget polynucleotide), where the copies comprise at least a portion ofthe template sequence and/or its complement. Such copies may besingle-stranded or double-stranded.

A “template” or “template nucleic acid” or “template polynucleotide”refers to a polynucleotide that comprises the polynucleotide sequence tobe amplified. In some embodiments, the polynucleotide sequence to beamplified is flanked by primer hybridization sites, such as ahybridization site for a 5′ primer (or the complement thereof) and ahybridization site for a 3′ primer (or the complement thereof). Atemplate may comprise RNA and/or DNA, and may be from a natural source,or be synthetic. Nonlimiting exemplary templates include genomic DNA,viral DNA, mitochondrial DNA, viral RNA, mRNA, tRNA, microRNA, plasmids,vectors, cosmids, artificial chromosomes, etc. Any polynucleotide thatmay be copied or amplified by a polymerase enzyme is considered atemplate.

“Domain” refers to a unit of a protein or protein complex, comprising apolypeptide subsequence, a complete polypeptide sequence, or a pluralityof polypeptide sequences where that unit has a defined function. Thefunction is understood to be broadly defined and can be ligand binding,catalytic activity, and/or can have a stabilizing effect on thestructure of the protein.

Residues “correspond” to each other where they occur at equivalentpositions in aligned amino acid sequences, such as family B thermophilicpolymerase sequences and/or a domain thereof, such as a uracil-bindingpocket, catalytic domain, or exonuclease domain. Corresponding positionscan be identified as positions that align with one another. Related orvariant polypeptides are aligned by any method in the art. Such methodstypically maximize matches, and include methods such as using manualalignments and by using any of the numerous alignment programs available(for example, BLASTP) and others known in the art. By aligning thesequences of polypeptides, one of skill in the art can identifycorresponding residues, using conserved and identical amino acidresidues as guides. In some embodiments, an amino acid of a polypeptideis considered to correspond to an amino acid in a disclosed sequencewhen the amino acid of the polypeptide is aligned with the amino acid inthe disclosed sequence upon alignment of the polypeptide with thedisclosed sequence to maximize identity and homology (e.g., whereconserved amino acids are aligned) using a standard alignment algorithm,such as the BLASTP algorithm with default scoring parameters (such as,for example, BLOSUM62 Matrix, Gap existence penalty 11, Gap extensionpenalty 1, and with default general parameters). As a non-limitingexample, with reference to the multiple sequence alignment shown inFIGS. 11A-C, amino acid residue 408 in SEQ ID NO: 9 corresponds topositions 410, 407, 407, 407, and 408 in SEQ ID NOs: 52, 57, 55, 56, and51, respectively (marked with an asterisk in FIG. 11A). As anothernon-limiting example, amino acid residue 762 in SEQ ID NO: 9 correspondsto positions 764, 761, 761, 761, and 762 in SEQ ID NOs: 52, 57, 55, 56,and 51, respectively (marked with a pound in FIG. 11B). As anothernon-limiting example, amino acid residue 36 in SEQ ID NO: 9 correspondsto position 36 in SEQ ID NOs: 52, 57, 55, 56, and 51 (marked with apercent in FIG. 11B). In some embodiments, corresponding positions canalso be identified using overlaid 3-D structures, where available, aspositions at which greater than 50% of the volume occupied by aspace-filling model of an amino acid in a first polypeptide is occupiedby the space-filling model of the corresponding amino acid in a secondpolypeptide.

“Identity” is measured by a score determined by comparing the amino acidsequences of the two polypeptides using the Bestfit program. Bestfituses the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981) to find the best segment ofsimilarity between two sequences. When using Bestfit to determinewhether a test amino acid sequence is, for instance, 95% identical to areference sequence according to the present disclosure, the parametersare set so that the percentage of identity is calculated over the fulllength of the test amino acid sequence, such that 95% of the amino acidsin the test amino acid sequence align with identical amino acids on thereference sequence.

“Sequence-non-specific DNA binding domain” or “DNA binding domain”refers to a protein domain that binds to DNA without significantsequence preference. In some embodiments, a DNA binding domain binds todouble-stranded DNA. Non-limiting exemplary DNA binding domains includeSso7d from Sulfolobus solfataricus, Sac7d, Sac7a, Sac7b, and Sac7e fromS. acidocaldarius, and Ssh7a and Ssh7b from Sulfolobus shibatae,Pae3192, Pae0384, and Ape3192, HMf family archaeal histone domains, andarchaeal PCNA homolog.

With reference to two polypeptides or two polypeptide domains, the term“fused” means that the two polypeptides or polypeptide domains arecontained in a single contiguous polypeptide sequence.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. In someembodiments, such a protein, e.g., a fusion protein, contains two ormore domains from unrelated proteins arranged to make a new functionalprotein.

“Error-correcting activity” of a polymerase or polymerase domain refersto the 3′ to 5′ exonuclease proofreading activity of a polymerasewhereby nucleotides that do not form Watson-Crick base pairs with thetemplate are removed from the 3′ end of an oligonucleotide, i.e., astrand being synthesized from a template, in a sequential manner.Examples of polymerases that have error-correcting activity includepolymerases from Pyrococcus furiosus, Thermococcus litoralis, andThermotoga maritima with wild-type exonuclease domains, and certainothers discussed herein.

“Sensitivity” as used herein, refers to the ability of a polymerase toamplify a target nucleic acid that is present at low copy number. Insome embodiments, low copy number refers to a target nucleic acid thatis present at fewer than 10,000 or fewer than 1,000 or fewer than 100 orfewer than 10 copies in the composition comprising the target nucleicacid and the polymerase.

“Specificity” as used herein, refers to the ability of a polymerase toamplify a target nucleic acid while producing fewer non-specificamplification byproducts, such as those resulting from primer-dimers.

As used herein the terms “hybridize”, “hybridizing”, “hybridization” andother related terms include hydrogen bonding between two differentnucleic acids, or between two different regions of a nucleic acid, toform a duplex nucleic acid. Hybridization can comprise Watson-Crick orHoogstein binding to form a duplex nucleic acid. The two differentnucleic acids, or the two different regions of a nucleic acid, may becomplementary, or partially complementary. The complementary basepairing can be the standard A-T or C-G base pairing, or can be otherforms of base-pairing interactions. Duplex nucleic acids can includemismatched base-paired nucleotides. Complementary nucleic acid strandsneed not hybridize with each other across their entire length.

In some embodiments, conditions that are suitable for nucleic acidhybridization and/or nucleic acid synthesis include parameters such assalts, buffers, pH, temperature, % GC content of the polynucleotide andprimers, and/or time. For example, conditions suitable for hybridizingnucleic acids (e.g., polynucleotides and primers) can includehybridization solutions having sodium salts, such as NaCl, sodiumcitrate and/or sodium phosphate. In some embodiments, a hybridizationsolution can be a stringent hybridization solution which can include anycombination of formamide (e.g., about 50%), 5X SSC (e.g., about 0.75 MNaCl and about 0.075 M sodium citrate), sodium phosphate (e.g., about 50mM at about pH 6.8), sodium pyrophosphate (e.g., about 0.1%), 5XDenhardt’s solution, SDS (e.g., about 0.1%), and/or dextran sulfate(e.g., about 10%). In some embodiments, hybridization and/or nucleicacid synthesis can be conducted at a temperature range of about 45-55°C., or about 55-65° C., or about 65-75° C.

In some embodiments, hybridization or nucleic acid synthesis conditionscan be conducted at a pH range of about 5-10, or about pH 6-9, or aboutpH 6.5-8, or about pH 6.5-7.

Thermal melting temperature (T_(m)) for nucleic acids can be atemperature at which half of the nucleic acid strands aredouble-stranded and half are single-stranded under a defined condition.In some embodiments, a defined condition can include ionic strength andpH in an aqueous reaction condition. A defined condition can bemodulated by altering the concentration of salts (e.g., sodium),temperature, pH, buffers, and/or formamide. Typically, the calculatedthermal melting temperature can be at about 5-30° C. below the T_(m), orabout 5-25° C. below the T_(m), or about 5-20° C. below the T_(m), orabout 5-15° C. below the T_(m), or about 5-10° C. below the T_(m).Methods for calculating a T_(m) are well known and can be found inSambrook (1989 in “Molecular Cloning: A Laboratory Manual”, 2^(nd)edition, volumes 1-3; Wetmur 1966, J. Mol. Biol., 31:349-370; Wetmur1991 Critical Reviews in Biochemistry and Molecular Biology,26:227-259). Other sources for calculating a T_(m) for hybridizing ordenaturing nucleic acids include OligoAnalyze (from Integrated DNATechnologies) and Primer3 (distributed by the Whitehead Institute forBiomedical Research).

Provided herein are thermophilic DNA polymerases comprising a family Bpolymerase N-terminal domain comprising a uracil-binding pocket in whicha proline is replaced with another amino acid, and a family B polymerasecatalytic domain in which a neutral amino acid residue is present at acertain position. Many types of Family B polymerases are described inRothwell and Watsman, Advances in Protein Chemistry 71:401-440 (2005).Examples of thermophilic Family B polymerases include those of thePyrococcus and Thermococcus genera, such as the Deep Vent polymerase andFamily B polymerases of P. furiosus, P. calidifontis, P. aerophilum, T.kodakarensis, T. gorgonarius, and Thermococcus sp. 9°N-7. Exemplarywild-type amino acid sequences for such thermophilic family Bpolymerases can be obtained from public databases such as NCBI GenBankor UniProt. Wild-type sequences include naturally-occurring variants ofthe amino acid sequences for such thermophilic family B polymerases canbe obtained from public databases such as NCBI GenBank or UniProt. Notethat in some cases, the sequences are annotated as containing inteins;the inteins are not present in the mature enzyme.

In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has an amino acid sequence in whichthe position corresponding to position 36 of SEQ ID NO: 1 is any aminoacid other than P. In some embodiments, the family B polymeraseN-terminal domain comprising a uracil-binding pocket has an amino acidsequence wherein the amino acid residue at the position of the aminoacid sequence that aligns to position 36 of SEQ ID NO: 1 is any aminoacid other than P. In some embodiments, the family B polymeraseN-terminal domain comprising a uracil-binding pocket has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to a sequence selected fromSEQ ID NOs: 115 to 121 and 162 to 168, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 115, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 116, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 117, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 118, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 119, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 120, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 121, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 162, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 163, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 164, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 165, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 166, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 167, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket has at least 80%, 85%, 90%, 95%, 98%,99%, or 100% identity to SEQ ID NO: 168, wherein the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P.

In some embodiments, the family B polymerase catalytic domain has anamino acid sequence in which the position corresponding to position 379of SEQ ID NO: 6 is a neutral amino acid residue. In some embodiments,the family B polymerase catalytic domain has an amino acid sequencewherein the amino acid residue at the position of the amino acidsequence that aligns to position 379 of SEQ ID NO: 6 is a neutral aminoacid residue. In some embodiments, the family B polymerase catalyticdomain has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to asequence selected from SEQ ID NOs: 6 to 10, 15 to 18, 25, 26, 33, 34,37, 38, 41, 42, and 45 to 48, wherein the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue. In someembodiments, the family B polymerase catalytic domain has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 7, wherein theposition corresponding to position 379 of SEQ ID NO: 6 is a neutralamino acid residue. In some embodiments, the family B polymerasecatalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to the catalytic domain of SEQ ID NO: 15, wherein the positioncorresponding to position 379 of SEQ ID NO: 6 is a neutral amino acidresidue. In some embodiments, the family B polymerase catalytic domainhas at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to thecatalytic domain of SEQ ID NO: 25, wherein the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue. In someembodiments, the family B polymerase catalytic domain has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the catalytic domain of SEQID NO: 33, wherein the position corresponding to position 379 of SEQ IDNO: 6 is a neutral amino acid residue. In some embodiments, the family Bpolymerase catalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%,or 100% identity to the catalytic domain of SEQ ID NO: 37, wherein theposition corresponding to position 379 of SEQ ID NO: 6 is a neutralamino acid residue. In some embodiments, the family B polymerasecatalytic domain has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to the catalytic domain of SEQ ID NO: 47, wherein the positioncorresponding to position 379 of SEQ ID NO: 6 is a neutral amino acidresidue. In some embodiments, the family B polymerase catalytic domainhas at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to thecatalytic domain of SEQ ID NO: 41, wherein the position corresponding toposition 379 of SEQ ID NO: 6 is a neutral amino acid residue. In someembodiments, the family B polymerase catalytic domain has at least 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the catalytic domain of SEQID NO: 45, wherein the position corresponding to position 379 of SEQ IDNO: 6 is a neutral amino acid residue.

Examples of family B polymerase catalytic domain sequences are shown,e.g., in FIG. 12 . In some embodiments, the C-terminus is the residue atthe position of the conserved leucine shown as the last residue in themultiple sequence alignment in FIG. 12 . In some embodiments, theC-terminus of the family B polymerase catalytic domain is the positioncorresponding to position 383 of SEQ ID NO: 6. In some embodiments, theC-terminus of the family B polymerase catalytic domain is the positioncorresponding to the leucine which is the last residue of SEQ ID NO: 6.In some embodiments, the C-terminus of the family B polymerase catalyticdomain is the position that aligns to the leucine which is the lastresidue of SEQ ID NO: 6. In some embodiments, the C-terminus of thefamily B polymerase catalytic domain is the position corresponding to aleucine selected from the leucines shown as the final residues in FIG.12 . In some embodiments, the C-terminus of the family B polymerasecatalytic domain is the position that aligns to a leucine selected fromthe leucines shown as the final residues in FIG. 12 . The C-terminalresidue in any of the foregoing embodiments can be a leucine.

In some embodiment, the thermophilic DNA polymerase comprises anN-terminal domain comprising a uracil-binding pocket that comprises: (a)the consecutive amino acid residues RX¹YIY (SEQ ID NO: 199), (b) theconsecutive amino acid residues QX¹YIY (SEQ ID NO: 200), (c) theconsecutive amino acid residues EX¹YIY (SEQ ID NO: 201), (d) theconsecutive amino acid residues EX¹YFY (SEQ ID NO: 202), or (e) theconsecutive amino acid residues RX¹YFY (SEQ ID NO: 203); wherein X¹ isany amino acid other than P; and wherein X¹ is within 50, 45, 44, 43,42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 residues of theN-terminus of the family B polymerase N-terminal domain comprising auracil-binding pocket. In some embodiments, the N-terminus of the familyB polymerase N-terminal domain comprising a uracil-binding pocket is theN-terminus of the thermophilic DNA polymerase. In some embodiments, X¹is within 42, 41, 40, 39, 38, 37, or 36 residues of the N-terminus ofthe family B polymerase N-terminal domain comprising a uracil-bindingpocket. In some embodiments, X¹ is within 42 residues of the N-terminusof the family B polymerase N-terminal domain comprising a uracil-bindingpocket. In some embodiments, X¹ is within 40 residues of the N-terminusof the family B polymerase N-terminal domain comprising a uracil-bindingpocket. In some embodiments, X¹ is within 36 residues of the N-terminusof the family B polymerase N-terminal domain comprising a uracil-bindingpocket.

In some embodiments, the thermophilic DNA polymerase comprises: (a) theconsecutive amino acid residues WQKTX² (SEQ ID NO: 204), (b) theconsecutive amino acid residues YQKTX² (SEQ ID NO: 205), (c) theconsecutive amino acid residues X²QTGL (SEQ ID NO: 206), (d) theconsecutive amino acid residues X²QVGL (SEQ ID NO: 207), (e) theconsecutive amino acid residues KTX²QT (SEQ ID NO: 208), or (f) theconsecutive amino acid residues KTX²QV (SEQ ID NO: 209); wherein X² is aneutral amino acid residue; and wherein X² is within 20, 15, 10, 5, or 4residues of the C-terminus of the family B polymerase catalytic domain.In some embodiments, The C-terminus of the family B polymerase catalyticdomain can be identified as the amino acid that aligns to or correspondsto the last amino acid of SEQ ID NO: 6. In some embodiments, thethermophilic DNA polymerase comprises a consecutive amino acid sequenceof WQKTX² (SEQ ID NO: 204), X²QTGL (SEQ ID NO: 206), KTX²QT (SEQ ID NO:208), YQKTX² (SEQ ID NO: 205), X²QVGL (SEQ ID NO: 207), KTX²QV (SEQ IDNO: 209), YQSSX² (SEQ ID NO: 210), X²QTGL (SEQ ID NO: 206), SSX²QT (SEQID NO: 211),; wherein X² is a neutral amino acid residue; and wherein X²is within 20, 15, 10, 5, or 4 residues of the C-terminus of the family Bpolymerase catalytic domain. In some embodiments, the thermophilic DNApolymerase comprises a consecutive amino acid sequence of WQKTX² (SEQ IDNO: 204), X²QTGL (SEQ ID NO: 206), KTX²QT (SEQ ID NO: 208), YQKTX² (SEQID NO: 205), X²QVGL (SEQ ID NO: 207), KTX²QV (SEQ ID NO: 209), YQSSX²(SEQ ID NO: 210), X²QTGL (SEQ ID NO: 206), SSX²QT (SEQ ID NO: 211),TGRVX² (SEQ ID NO: 212), X²KSLL (SEQ ID NO: 213), RVX²KS (SEQ ID NO:214), TGRSX² (SEQ ID NO: 215), X²RTLL (SEQ ID NO: 216), or RSX²RT (SEQID NO: 217); wherein X² is a neutral amino acid residue; and wherein X²is within 20, 15, 10, 5, or 4 residues of the C-terminus of the family Bpolymerase catalytic domain. X² can be within 15 residues of theC-terminus of the family B polymerase catalytic domain in any of theforegoing embodiments. X² can be within 10 residues of the C-terminus ofthe family B polymerase catalytic domain in any of the foregoingembodiments. X² can be within 5 residues of the C-terminus of the familyB polymerase catalytic domain in any of the foregoing embodiments. X²can be within 4 residues of the C-terminus of the family B polymerasecatalytic domain in any of the foregoing embodiments. For the avoidanceof doubt, in a sequence segment consisting of n residues, residues 1 ton are within n-1 residues of position n; e.g., if n is 5, positions 1,2, 3, 4, and 5 and are within 4 residues of position 5.

This paragraph concerns the neutral amino acid residue referred to inany of the embodiments mentioned in the preceding paragraphs. Neutralamino acid residues do not have side chains containing groups that aremore than 50% charged at pH 7.4 in aqueous solution at 37° C., such ascarboxyls, amines, and guanidino groups. Neutral amino acid residuesinclude canonical and noncanonical residues unless indicated to thecontrary. In some embodiments, the neutral amino acid is a noncanonicalresidue. A noncanonical residue is a residue other than the twenty aminoacid residues abbreviated as one of the twenty following letters: A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y (e.g., norleucineand selenomethionine are noncanonical; see, e.g., U.S. Pat. No.7,541,170 for additional examples of noncanonical residues, which arereferred to therein as “nonclassical amino acids or chemical amino acidanalogs”). In some embodiments, the neutral amino acid is less than 10%,1%, 0.1%, or 0.01% charged at pH 7.4 in aqueous solution at 37° C. Insome embodiments, the neutral amino acid residue is a polar neutralamino acid residue. A residue is polar if its side chain contains atleast one hydrogen bond donor or acceptor. In some embodiments, theneutral amino acid comprises a side chain comprising an alcohol, amide,carbonyl, ester, or ether. In some embodiments, the neutral amino acidcomprises a side chain comprising an alcohol. In some embodiments, theneutral amino acid comprises a side chain comprising an amide. In someembodiments, the neutral amino acid residue is Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, or G. In some embodiments, the neutral amino acidresidue is Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, or G. In someembodiments, the neutral amino acid residue is Q, N, S, T, C, M, A, I,L, V, or G. In some embodiments, the neutral amino acid residue is Q, N,S, T, C, M, A, or G. In some embodiments, the neutral amino acid residueis Q, N, H, S, T, Y, C, M, or W. In some embodiments, the neutral aminoacid residue is Q, N, H, S, T, Y, or W. In some embodiments, the neutralamino acid residue is Q, N, H, S, T, C, or M. In some embodiments, theneutral amino acid residue is Q, N, S, T, C, or M. In some embodiments,the neutral amino acid residue is Q, N, S, or T. In some embodiments,the neutral amino acid residue is Q or N. In some embodiments, theneutral amino acid residue is S. In some embodiments, the neutral aminoacid residue is T. In some embodiments, the neutral amino acid residueis Q. In some embodiments, the neutral amino acid residue is N.

In some embodiments, the family B polymerase catalytic domain is asubfamily B3 polymerase domain. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain of aPyrococcus in which a neutral amino acid residue is present at aposition discussed above. In some embodiments, the family B polymerasecatalytic domain is a family B polymerase domain of a Thermococcus inwhich a neutral amino acid residue is present at a position discussedabove. In some embodiments, the family B polymerase catalytic domain isa family B polymerase domain of a Pyrobaculum in which a neutral aminoacid residue is present at a position discussed above. In someembodiments, the family B polymerase catalytic domain is a family Bpolymerase domain of Pyrococcus furiosus in which a neutral amino acidresidue is present at a position discussed above. In some embodiments,the family B polymerase catalytic domain is a family B polymerase domainof Pyrococcus species GB-D in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus kodakarensis in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus litoralis in which a neutral amino acid residue is presentat a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus gorgonarius in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofThermococcus sp. 9°N-7 in which a neutral amino acid residue is presentat a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofPyrobaculum calidifontis in which a neutral amino acid residue ispresent at a position discussed above. In some embodiments, the family Bpolymerase catalytic domain is a family B polymerase domain ofPyrobaculum aerophilum in which a neutral amino acid residue is presentat a position discussed above.

In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket is a subfamily B3 N-terminal domain.In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket is a family B N-terminal domain of aPyrococcus in which the position corresponding to position 36 of SEQ IDNO: 1 is any amino acid other than P. In some embodiments, the family Bpolymerase N-terminal domain comprising a uracil-binding pocket is afamily B N-terminal domain of a Thermococcus in which the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket is a family B N-terminal domain of aPyrobaculum in which the position corresponding to position 36 of SEQ IDNO: 1 is any amino acid other than P. In some embodiments, the family Bpolymerase N-terminal domain comprising a uracil-binding pocket is afamily B N-terminal domain of Pyrococcus furiosus in which the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket is a family B N-terminal domain ofPyrococcus species GB-D in which the position corresponding to position36 of SEQ ID NO: 1 is any amino acid other than P. In some embodiments,the family B polymerase N-terminal domain comprising a uracil-bindingpocket is a family B N-terminal domain of Thermococcus kodakarensis inwhich the position corresponding to position 36 of SEQ ID NO: 1 is anyamino acid other than P. In some embodiments, the family B polymeraseN-terminal domain comprising a uracil-binding pocket is a family BN-terminal domain of Thermococcus litoralis in which the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket is a family B N-terminal domain ofThermococcus gorgonarius in which the position corresponding to position36 of SEQ ID NO: 1 is any amino acid other than P. In some embodiments,the family B polymerase N-terminal domain comprising a uracil-bindingpocket is a family B N-terminal domain of Thermococcus sp. 9°N-7 inwhich the position corresponding to position 36 of SEQ ID NO: 1 is anyamino acid other than P. In some embodiments, the family B polymeraseN-terminal domain comprising a uracil-binding pocket is a family BN-terminal domain of Pyrobaculum calidifontis in which the positioncorresponding to position 36 of SEQ ID NO: 1 is any amino acid otherthan P. In some embodiments, the family B polymerase N-terminal domaincomprising a uracil-binding pocket is a family B N-terminal domain ofPyrobaculum aerophilum in which the position corresponding to position36 of SEQ ID NO: 1 is any amino acid other than P.

In some embodiments, all domains of the thermophilic DNA polymerase arecontained in a single polypeptide. In some embodiments, the thermophilicDNA polymerase comprises a plurality of polypeptide chains, which may benoncovalently associated or covalently associated. In some embodiments,the plurality of polypeptide chains can include a first polypeptidecomprising an N-terminal domain comprising a uracil-binding procket anda polymerase catalytic domain and a second polypeptide comprising anadditional domain, such as a sequence non-specific double-strandedDNA-binding domain. A covalent association can include, e.g., one ormore disulfide bonds or chemical conjugation using a linking compound,e.g., a chemical crosslinking agent, including, for example,succinimidyl-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC).Disulfide bonds and chemical conjugation are discussed further below.

In some embodiments, the thermophilic DNA polymerase comprises asequence non-specific DNA-binding domain, e.g., a thermostable DNAbinding domain. The DNA binding domain can be, for example, present aspart of a fusion protein with the polymerase catalytic domain. In someembodiments, the DNA binding domain is fused C-terminal to thepolymerase catalytic domain. In some embodiments, the DNA binding domainis noncovalently associated with the polypeptide comprising thepolymerase catalytic domain, e.g., in the manner of the associationbetween sliding clamps and certain family B polymerases. In someembodiments, the polypeptide comprising the polymerase catalytic domainfurther comprises a sequence that noncovalently associates with an DNAbinding domain, such as the PCNA-interacting sequence of a dimericarchaeal polymerase such as Pfu Pol II. As discussed, e.g., in U.S. Pat.No. 7,541,170, an DNA binding domain can provide improved processivityrelative to version of the enzyme lacking the DNA binding domain.Processivity reflects the extent to which a polymerase continues tosynthesize DNA (adding nucleotides in processive catalytic events) alongthe same template without falling off. In some embodiments, highprocessivity correlates to high sensitivity in amplification reactions.

In some embodiments, the DNA binding domain is covalently conjugated tothe polypeptide comprising the polymerase catalytic domain. Techniquesfor covalent conjugation of heterologous domains are described, e.g., inBIOCONJUGATE TECHNIQUES, Hermanson, Ed., Academic Press (1996). Suchtechniques include, for example, derivitization for the purpose oflinking the moieties to each other, either directly or through a linkingcompound, by methods that are well known in the art of proteinchemistry. For example, in one chemical conjugation embodiment, thecatalytic domain and the nucleic acid binding domain are linked using aheterobifunctional coupling reagent which ultimately contributes toformation of an intermolecular disulfide bond between the two moieties.Other types of coupling reagents that are useful in this capacity forthe present invention are described, for example, in U.S. Pat. No.4,545,985. Alternatively, an intermolecular disulfide may convenientlybe formed between cysteines in each moiety, which occur naturally or areinserted by genetic engineering. The means of linking moieties may alsouse thioether linkages between heterobifunctional crosslinking reagentsor specific low pH cleavable crosslinkers or specific protease cleavablelinkers or other cleavable or noncleavable chemical linkages.

In some embodiments, the sequence non-specific double-strandedDNA-binding domain comprises an amino acid sequence having at least 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to an amino acid sequenceselected from SEQ ID NOs: 53 to 62. In some embodiments, the DNA bindingdomain is an archaeal DNA binding domain. In some embodiments, the DNAbinding domain is a 7kD DNA-binding domain, which occurs in certainarchaeal small basic DNA binding proteins (see, e.g., Choli et al.,Biochimica et Biophysica Acta 950:193-203, 1988; Baumann et al.,Structural Biol. 1:808-819, 1994; and Gao et al, Nature Struc. Biol.5:782-786, 1998). Additional archaeal DNA binding domains are discussedin Hardy and Martin, Extremophiles 12:235-46 (2008).

In some embodiments, the DNA binding domain is an Sso7d domain. In someembodiments, the DNA binding domain is a Sac7d domain. In someembodiments, the DNA binding domain is a Sac7e domain. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 53. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 54. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 62.

In some embodiments, the DNA binding domain is a Pae3192 domain. In someembodiments, the DNA binding domain is a Pae0384 domain. In someembodiments, the DNA binding domain is a Ape3192 domain. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 55. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 56. In someembodiments, the sequence non-specific double-stranded DNA-bindingdomain comprises an amino acid sequence having at least 75%, 80%, 85%,90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 57.

In some embodiments, the DNA binding domain is an archaeal histonedomain. In some embodiments, the archaeal histone domain is an HMffamily archaeal histone domain (see, e.g., Starich et al., J Molec.Biol. 255:187-203, 1996; Sandman et al., Gene 150:207-208, 1994). Insome embodiments, the archaeal histone domain is an HMf family archaealhistone domain from Methanothermus. In some embodiments, the archaealhistone domain is an HMf family archaeal histone domain from Pyrococcus.In some embodiments, the archaeal histone domain is an HMf familyarchaeal histone domain from Methanothermus fervidus. In someembodiments, the archaeal histone domain is an HMf family archaealhistone domain from Pyrococcus strain GB-3a. In some embodiments, thearchaeal histone domain is a Methanothermus HMfA archaeal histonedomain. In some embodiments, the archaeal histone domain is aMethanothermus HMfB archaeal histone domain. In some embodiments, thearchaeal histone domain is a Pyrococcus HpyA1 archaeal histone domain.In some embodiments, the archaeal histone domain is a Pyrococcus HpyA2archaeal histone domain. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 58. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 59. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 60. In some embodiments, the sequence non-specificdouble-stranded DNA-binding domain comprises an amino acid sequencehaving at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity toSEQ ID NO: 61.

In some embodiments, the DNA binding domain is a sliding clamp, such asan archaeal PCNA homolog. Sliding clamps can exist as trimers insolution, and can form a ring-like structure with a central passagecapable of accommodating double-stranded DNA. The sliding clamp formsspecific interactions with the amino acids located at the C terminus ofparticular DNA polymerases, and tethers those polymerases to the DNAtemplate during replication. The sliding clamp in eukaryotes is referredto as the proliferating cell nuclear antigen (PCNA), while similarproteins in other domains are often referred to as PCNA homologs. Thesehomologs have marked structural similarity but limited sequencesimilarity. PCNA homologs have been identified from thermophilic Archaea(e.g., Sulfolobus solfataricus, Pyrococcus furiosus, etc.). Some familyB polymerases in Archaea have a C terminus containing a consensusPCNA-interacting amino acid sequence and are capable of using a PCNAhomolog as a processivity factor (see, e.g., Cann et al., J. Bacteriol.181:6591-6599, 1999 and De Felice et al., J Mol. Biol. 291:47-57, 1999).These PCNA homologs are useful sequence-non-specific double-stranded DNAbinding domains. For example, a consensus PCNA-interacting sequence canbe joined to a polymerase that does not naturally interact with a PCNAhomolog, thereby allowing a PCNA homolog to serve as a processivityfactor for the polymerase. By way of illustration, the PCNA-interactingsequence from Pyrococcus furiosus Pol II (a heterodimeric DNA polymerasecontaining two family B-like polypeptides) can be covalently joined to asequence based on Pyrococcus furiosus Pol I (a monomeric family Bpolymerase that does not normally interact with a PCNA homolog). Theresulting fusion protein can then be allowed to associate noncovalentlywith the Pyrococcus furiosus PCNA homolog to generate a heterologousprotein with increased processivity.

Nucleic acids encoding the domains of a fusion protein invention can beobtained using recombinant genetics techniques. Basic texts disclosingthe general methods for doing so include Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL (2nd ed. 1989); Kriegler, GENE TRANSFER ANDEXPRESSION:A LABORATORY MANUAL (1990); and CURRENT PROTOCOLS INMOLECULAR BIOLOGY (Ausubel et al., eds., 1994)).

In some embodiments, catalytic and binding domains of the polymerase arejoined by a linker domain, e.g., a polypeptide sequence of 1 to about200 amino acids in length, such as 1 to about 100, 50, 25, or 10 aminoacids. In some embodiments, proline residues are incorporated into thelinker to prevent the formation of significant secondary structuralelements by the linker. Linkers can often be flexible amino acidsubsequences that are synthesized as part of a recombinant fusionprotein. For a discussion of linkers, see, e.g., US 2011/0086406 A1including at paragraphs 83-89 thereof.

In some embodiments, the thermophilic DNA polymerase comprises anexonuclease domain. In some embodiments, the exonuclease domain is a 3′to 5′ exonuclease domain. The 3′-5′ exonuclease domain can haveerror-correcting activity, also known as proofreading activity, in whichthe exonuclease preferentially removes a base from a nascent DNAstrand/extension product/3′ terminus that is not a Watson-Crick match tothe template strand. In some embodiments, the 3′-5′ exonuclease domainis a DEDDy archaeal exonuclease domain. In some embodiments, theexonuclease domain is N-terminal to the DNA polymerase catalytic domain.In some embodiments, the exonuclease domain is C-terminal to theN-terminal domain comprising a uracil-binding pocket. In someembodiments, the exonuclease domain is N-terminal to the DNA polymerasecatalytic domain and C-terminal to the N-terminal domain comprising auracil-binding pocket. In some embodiments, the thermophilic DNApolymerase comprises a domain having at least 75%, 80%, 85%, 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 63. In some embodiments, thethermophilic DNA polymerase comprises a domain having at least 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domain of asequence selected from SEQ ID NO: 1, 19, 23, 31, 35, 39, 43, 49, 51, or52. In some embodiments, the thermophilic DNA polymerase comprises adomain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%identity to the exonuclease domain of SEQ ID NO: 1. In some embodiments,the thermophilic DNA polymerase comprises a domain having at least 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domainof SEQ ID NO: 19. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of SEQ ID NO: 23. In someembodiments, the thermophilic DNA polymerase comprises a domain havingat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to theexonuclease domain of SEQ ID NO: 31. In some embodiments, thethermophilic DNA polymerase comprises a domain having at least 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domain ofSEQ ID NO: 35. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of SEQ ID NO: 39. In someembodiments, the thermophilic DNA polymerase comprises a domain havingat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to theexonuclease domain of SEQ ID NO: 43. In some embodiments, thethermophilic DNA polymerase comprises a domain having at least 75%, 80%,85%, 90%, 95%, 98%, 99%, or 100% identity to the exonuclease domain ofSEQ ID NO: 49. In some embodiments, the thermophilic DNA polymerasecomprises a domain having at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or100% identity to the exonuclease domain of SEQ ID NO: 51. In someembodiments, the thermophilic DNA polymerase comprises a domain havingat least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to theexonuclease domain of SEQ ID NO: 52. An exonuclease domain can beidentified using BLASTP against the RefSeq database can be identified byusing NCBI BLASTP to search the RefSeq database. NCBI BLASTPautomatically identifies certain domains such as exonuclease domains andindicates their termini as the positions at which the domain begins andends.

In some embodiments, the exonuclease domain is an exonuclease domain ofa Pyrococcus. In some embodiments, the exonuclease domain is anexonuclease domain of a Thermococcus. In some embodiments, theexonuclease domain is an exonuclease domain of a Pyrobaculum. In someembodiments, the exonuclease domain is an exonuclease domain ofPyrococcus furiosus. In some embodiments, the exonuclease domain is anexonuclease domain of Pyrococcus species GB-D. In some embodiments, theexonuclease domain is an exonuclease domain of Thermococcuskodakarensis. In some embodiments, the exonuclease domain is anexonuclease domain of Thermococcus litoralis. In some embodiments, theexonuclease domain is an exonuclease domain of Thermococcus gorgonarius.In some embodiments, the exonuclease domain is an exonuclease domain ofThermococcus sp. 9°N-7. In some embodiments, the exonuclease domain isan exonuclease domain of Pyrobaculum calidifontis. In some embodiments,the exonuclease domain is an exonuclease domain of Pyrobaculumaerophilum.

In some embodiments, the thermophilic DNA polymerase comprises aninactivated or reduced-activity exonuclease domain. An inactivatedexonuclease domain is a mutated version of a wild-type domain that hasless than 50% of the wild-type exonuclease activity. In someembodiments, the inactivated domain has less than 40%, less than 30%,less than 25%, less than 20%, less than 15%, less than 10%, or less than5% of the wild-type exonuclease activity. In some embodiments, theinactivated domain has less than 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%,or 0.01% of the wild-type exonuclease activity. A reduced-activityexonuclease domain is a mutated version of a wild-type domain that hasless than 10% of the wild-type exonuclease activity. Measurement ofexonuclease activity is described, for example, in DNA Replication 2nd,edition, by Kornberg and Baker, W.H. Freeman & Company, New York, N.Y.1991. Examples of exo⁻ DNA polymerase mutants include those with asingle mutation in Motif I and/or II (Motifs are as described, e.g., inU.S. Pat. No. 8,921,043, e.g., at FIG. 2 ), or a double mutation inMotif I (such as D141A and E143A, the position numbering corresponds toPfu polymerase, SEQ ID NO: 1), that reportedly abolishes detectibleexonuclease activity (see for example, VENT® (Thermococcus litoralis)(Kong et al. J. Biol. Chem. 268(3):1965-1975) (New England Biolabs, Inc.(NEB), Ipswich, Mass.); Thermococcus JDF-3 (U.S. Pat. No. 6,946,273,U.S. 2005/0069908); KODI (Thermococcus kodakaraensis) (U.S. Pat. No.6,008,025); Pfu (Pyrococcus furiosus) (U.S. Pat. No. 5,489,523, U.S.Pat. No. 7,704,712, and U.S. Pat. No. 7,659,100); and 9° N (Thermococcussp.) (U.S. 2005/0123940 and Southworth et al. Proc Natl Acad Sci USA93:5281-5285 (1996)); see also U.S. Pat. No. 8,921,043. In someembodiments, the exonuclease domain has a D141A, E143A, D215A, D315A,D141A/E143A, D141A/D315A, E143A/D315A, D215A/D315A, or D141A/E143A/D315Amutation. In some embodiments, the exonuclease domain has an A, N, S, T,or E residue at the position corresponding to position 141 of SEQ IDNO: 1. In some embodiments, the exonuclease domain has an A at theposition corresponding to position 141 of SEQ ID NO: 1. In someembodiments, the exonuclease domain has an A at the positioncorresponding to position 143 of SEQ ID NO: 1.

In some embodiments, the amino acid residue at the position of thefamily B polymerase catalytic domain amino acid sequence that aligns toposition 25 of SEQ ID NO: 6 is a serine. In some embodiments, the aminoacid residue at the position of the family B polymerase catalytic domainamino acid sequence that corresponds to position 25 of SEQ ID NO: 6 is aserine. In some embodiments, the thermophilic DNA polymerase comprises:(a) the consecutive amino acid residues LDFRS (SEQ ID NO: 196), (b) theconsecutive amino acid residues FRSLY (SEQ ID NO: 197), or (c) theconsecutive amino acid residues SLYPS (SEQ ID NO: 198), wherein theunderlined serine residue is within 30 amino acid residues of theN-terminus of the family B polymerase catalytic domain. In someembodiments, the thermophilic DNA polymerase comprises:

(a) the consecutive amino acid residues LDFRS* (SEQ ID NO: 196), (b) theconsecutive amino acid residues FRS*LY (SEQ ID NO: 197), or (c) theconsecutive amino acid residues S*LYPS (SEQ ID NO: 198), wherein theserine residue immediately followed by an asterisk is within 30 aminoacid residues of the N-terminus of the family B polymerase catalyticdomain. The asterisk is included solely to designate the serine that iswithin 30 amino acid residues of the N-terminus of the family Bpolymerase catalytic domain and does not signify a structuraldifference. In some embodiments, the N-terminus of the family Bpolymerase catalytic domain is the residue immediately preceding theconserved tyrosine shown as the second residue in the multiple sequencealignment in FIG. 12 . In some embodiments, the N-terminus of the familyB polymerase catalytic domain is the position immediately preceding theposition corresponding to the first tyrosine in SEQ ID NO: 6. In someembodiments, the N-terminus of the family B polymerase catalytic domainis the position that aligns to position 1 of SEQ ID NO: 6. In someembodiments, the N-terminus of the family B polymerase catalytic domainis the position corresponding to the position immediately preceding atyrosine selected from the tyrosines shown as the second residues inFIG. 12 . In some embodiments, the N-terminus of the family B polymerasecatalytic domain is the position immediately preceding the position thataligns to a tyrosine selected from the tyrosines shown as the secondresidues in FIG. 12 . In some embodiments, the N-terminus of the familyB polymerase catalytic domain is the position immediately preceding theposition corresponding to a tyrosine selected from the tyrosines shownas the second residues in FIG. 12 . The N-terminal residue in any of theforegoing embodiments can be a serine. The N-terminal residue in any ofthe foregoing embodiments can be a threonine. The N-terminal residue inany of the foregoing embodiments can be a glycine. The N-terminalresidue in any of the foregoing embodiments can be a proline.

As will be apparent from various aspects of the discussion above, familyB polymerases are well-characterized in general and are known totolerate mutations at a number of positions. Furthermore, the followingis a non-exhaustive list of patents and published applications thatdiscuss mutations in family B polymerases and the properties of mutatedfamily B polymerases: U.S. Pat. 8,435,775; U.S. Pat. 8,557,554;WO2007/016702; US 2003/0180741; WO 2004/011605; WO 2003/060144; and U.S.Pat. 9,023,633. Accordingly, those skilled in the art will understand inview of this disclosure that the residues discussed herein such as theneutral amino acid at the position corresponding to position 379 of SEQID NO: 6 can be incorporated into a wide variety of thermophilic familyB polymerases and can be accompanied by other amino acid residues thatdiffer from wild-type residues. Thus, in some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence comprisingat least one difference from SEQ ID NO: 1 at a position corresponding toposition 15, 72, 93, 141, 143, 247, 265, 337, 385, 387, 388, 399, 400,405, 407, 410, 485, 542, 546, 593, or 595 of SEQ ID NO: 1. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence comprising at least one missing residue corresponding toposition 92, 93, 94, or 381 of SEQ ID NO: 1. In some embodiments, the atleast one difference or missing residue is in the exonuclease domain. Insome embodiments, the at least one difference or missing residue is inthe polymerase catalytic domain.

In some embodiments, the polymerase with the at least one difference ormissing residue has an expanded substrate range relative to a polymerasewithout the difference or in which the residue is not missing. In someembodiments, the at least one difference comprises a G or D at theposition corresponding to position 400 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises an I at the positioncorresponding to position 407 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises an I at the position corresponding toposition 337 of SEQ ID NO: 1. In some embodiments, the at least onedifference comprises a D at the position corresponding to position 399of SEQ ID NO: 1. In some embodiments, the at least one differencecomprises an H at the position corresponding to position 546 of SEQ IDNO: 91.

In some embodiments, the polymerase with the at least one difference ormissing residue incorporates a nucleotide analog to a greater extentthan a polymerase without the difference or in which the residue is notmissing. In some embodiments, the at least one difference comprises an Lat the position corresponding to position 410 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises a T at the positioncorresponding to position 485 of SEQ ID NO: 1.

In some embodiments, the polymerase with the at least one difference ormissing residue has reduced uracil sensitivity relative to a polymerasewithout the difference or in which the residue is not missing. In someembodiments, the at least one missing residue comprises a missingresidue at the position corresponding to position 93 of SEQ ID NO: 1. Insome embodiments, the at least one missing residue comprises a missingresidue at the position corresponding to position 94 of SEQ ID NO: 1. Insome embodiments, the at least one missing residue comprises a missingresidue at the position corresponding to position 92 of SEQ ID NO: 1. Insome embodiments, the at least one difference comprises a Q, R, E, A, K,N, or G at the position corresponding to position 93 of SEQ ID NO: 1. Insome embodiments, the at least one difference comprises a Q or R at theposition corresponding to position 93 of SEQ ID NO: 1. In someembodiments, an at least one difference or missing residue as discussedabove in this paragraph is accompanied by at least one difference ormissing residue that offsets a loss of activity. In some embodiments,the at least one difference that offsets a loss of activity comprises anR at the position corresponding to position 247 of SEQ ID NO: 1. In someembodiments, the at least one difference that offsets a loss of activitycomprises an R at the position corresponding to position 265 of SEQ IDNO: 1. In some embodiments, the at least one difference that offsets aloss of activity comprises an R at the position corresponding toposition 485 of SEQ ID NO: 1. In some embodiments, the at least onemissing residue that offsets a loss of activity comprises a missingresidue at the position corresponding to position 381 of SEQ ID NO: 1.

In some embodiments, the at least one difference comprises an R at theposition corresponding to position 247 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises an R at the positioncorresponding to position 265 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises an R at the position corresponding toposition 485 of SEQ ID NO: 1. In some embodiments, the at least onemissing residue comprises a missing residue at the positioncorresponding to position 381 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises an I at the position corresponding toposition 15 of SEQ ID NO: 1. In some embodiments, the at least onedifference comprises an R at the position corresponding to position 72of SEQ ID NO: 1.

In some embodiments, the polymerase with the at least one difference ormissing residue has an altered proofreading spectrum relative to apolymerase without the difference or in which the residue is notmissing. In some embodiments, the at least one difference comprises a Por S at the position corresponding to position 387 of SEQ ID NO: 1. Insome embodiments, the at least one difference comprises an E at theposition corresponding to position 405 of SEQ ID NO: 1. In someembodiments, the at least one difference comprises an F at the positioncorresponding to position 410 of SEQ ID NO: 1. In some embodiments, theat least one difference comprises a P at the position corresponding toposition 542 of SEQ ID NO: 1. In some embodiments, the at least onedifference comprises a T at the position corresponding to position 593of SEQ ID NO: 1. In some embodiments, the at least one differencecomprises an S at the position corresponding to position 595 of SEQ IDNO: 1. In some embodiments, the at least one difference comprises a Q,S, N, L, or H at the position corresponding to position 385 of SEQ IDNO: 1. In some embodiments, the at least one difference comprises a P atthe position corresponding to position 388 of SEQ ID NO: 1.

In some embodiments, the thermophilic DNA polymerase comprises an aminoacid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to asequence selected from SEQ ID NOs: 11 to 14, 19 to 22, 27 to 30, and 76to 79. In some embodiments, the thermophilic DNA polymerase comprises anamino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identityto SEQ ID NO: 11. In some embodiments, the thermophilic DNA polymerasecomprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or100% identity to SEQ ID NO: 12. In some embodiments, the thermophilicDNA polymerase comprises an amino acid sequence having at least 90%,95%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments,the thermophilic DNA polymerase comprises an amino acid sequence havingat least 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 14. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ IDNO: 19. In some embodiments, the thermophilic DNA polymerase comprisesan amino acid sequence having at least 90%, 95%, 98%, 99%, or 100%identity to SEQ ID NO: 20. In some embodiments, the thermophilic DNApolymerase comprises an amino acid sequence having at least 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence having atleast 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 22. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ IDNO: 27. In some embodiments, the thermophilic DNA polymerase comprisesan amino acid sequence having at least 90%, 95%, 98%, 99%, or 100%identity to SEQ ID NO: 28. In some embodiments, the thermophilic DNApolymerase comprises an amino acid sequence having at least 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 29. In some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence having atleast 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 30. In someembodiments, the thermophilic DNA polymerase comprises an amino acidsequence having at least 90%, 95%, 98%, 99%, or 100% identity to SEQ IDNO: 76. In some embodiments, the thermophilic DNA polymerase comprisesan amino acid sequence having at least 90%, 95%, 98%, 99%, or 100%identity to SEQ ID NO: 77. In some embodiments, the thermophilic DNApolymerase comprises an amino acid sequence having at least 90%, 95%,98%, 99%, or 100% identity to SEQ ID NO: 78. In some embodiments, thethermophilic DNA polymerase comprises an amino acid sequence having atleast 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 79.

In some embodiments, the polymerase comprises an affinity purificationtag. In some embodiments, the affinity purification tag comprises asequence of histidines, such as 6, 7, 8, 9, or 10 consecutive histidines(SEQ ID NO: 218). The affinity purification tag can be located, e.g., atthe N or C terminus of a polypeptide of the polymerase.

Hot Start Enzymes and Compositions

In some embodiments, a polymerase according to this disclosure isprovided as a hot-start enzyme or a hot start composition. Fordiscussion of hot-start enzymes and/or compositions, see, e.g., U.S.Pat. 5,338,671; U.S. Pat. 7,074,556; U.S. Publication 2015/0044683; U.S.Publication 2014/0099644. As used herein, the term “hot start” generallyrefers to a means of limiting the availability of an essential reactioncomponent (e.g., a polymerase) when the reaction mixture is maintainedat a first temperature (typically a lower temperature) until a secondtemperature (typically a higher temperature) is reached which allows theessential component to participate in the reaction. Hot start reactionstypically involve incubation at a first (e.g., lower) temperature andsubsequent elevation to a second (e.g., higher) temperature which allowsthe desired reaction to take place. Activation of the hot start reactionis preferably achieved by an incubation at a temperature which is equalto or higher than the primer hybridization (annealing) temperature usedin the amplification reaction to ensure primer binding specificity. Thelength of incubation required to recover enzyme activity depends on thetemperature and pH of the reaction mixture and on the stability of theenzyme. A wide range of incubation conditions are usable; optimalconditions may be determined empirically for each reaction.

As used herein, the term “dual hot start reaction mixture” refers to thecombination of reagents or reagent solutions which are used to blocknucleic acid polymerase extension at low temperatures (e.g., ambienttemperature) until the hot start conditions of the initial denaturationtemperature in an amplification reaction (e.g., PCR) are reached. At theelevated amplification temperature, the nucleic acid polymerase is nolonger inhibited and allows for primer extension. As used herein, thedual hot start reaction mixture is meant to include a reaction mixturethat comprises at least two different mechanisms for hot start.Accordingly, “dual hot start reaction mixtures” may include more thantwo hot start mechanisms (e.g., “triple hot start reaction mixture”,“quadruple hot start reaction mixture”, “quintuple hot start reactionmixture”, and so on).

Nonlimiting exemplary hot start mechanisms include, but are not limitedto, antibodies or combinations of antibodies that block nucleic acidpolymerase activity at lower temperatures and which dissociate from thepolymerase at elevated temperatures (see, e.g., Eastlund et al.,LifeSci. Quarterly 2:2 (2001), Mizuguchi et al., J. Biochem. (Tokyo)126:762 (1999)); affibodies (small synthetic protein molecules that havehigh binding affinity for a target protein) or combinantions ofaffibodies, sometimes referred to as antibody mimetics; oligonucleotidesthat block nucleic acid polymerase activity at lower temperatures andwhich dissociate from the polymerase at elevated temperatures (see,e.g., Dang et al., J. Mol. Biol. 264:268 (1996)); reversible chemicalmodification of the nucleic acid polymerase such that the nucleic acidpolymerase activity is blocked at lower temperatures and themodifications reverse or dissociate at elevated temperatures (see, e.g.,U.S. Pat. No. 5,773,258 and Moretti et al., Biotechniques 25:716(1998)); amino acid mutations of the nucleic acid polymerase thatprovide reduced activity at lower temperatures (see, e.g., Kermekchievet al., Nucl. Acids Res. 31:6139 (2003)); nucleic acid polymerase fusionproteins including hyperstable DNA binding domains and topoisomerases(see, e.g., Pavlov et al., Proc. Natl. Acad. Sci. USA 99:13510 (2002));ligands that inhibit the nucleic acid polymerase in atemperature-dependent manner (for example, HotMaster™ Taq DNA polymerasefrom Eppendorf (Hauppauge, N.Y.) and 5 PRIME (Gaithersburg, Md.));single-stranded binding proteins that sequester primers at lowtemperatures (see, e.g., U.S. Pat. Application Publication No.2008/0138878); thermostable pyrophosphatase which hydrolyzes inorganicpyrophosphate at elevated temperatures (see, e.g., U.S. Pat. ApplicationPublication No. 2006/0057617); thermolabile blockers, such as apolymerase blocking protein (see, e.g., U.S. Pat. ApplicationPublication No. 2007/0009922); primer competitor sequences (see, e.g.,Puskas et al., Genome Res. 5:309 (1995) and Vestheim et al., Front.Zool. 5:12 (2008)); modified primer constructs (see, e.g., Ailenberg etal., Biotechniques 29:22 (2000) and Kaboev et al., Nucl. Acids Res.28:E94 (2000)); modified primers that improve hybridization selectivity(see, e.g., U.S. Pat. Nos. 6,794,142 and 6,001,611); primers with 3′modifications that are removable by 3′-5′ exonuclease activity (see,e.g., U.S. Pat. Application Publication No. 2003/0119150 and U.S. Pat.No. 6,482,590); primers with modified nucleobases that are removable byUV irradiation (see, e.g., Young et al., Chem. Commun. (Camb) 28:462(2008)); primer modifications that are removable by thermal deprotection(see, e.g., U.S. Pat. Application Publication No. 2003/0162199 andLebedev et al., Nucl. Acids Res. 36:e131 (2008)); or modification of thedNTPs with thermolabile modification groups (see, e.g., U.S. Pat.Application Publication No. 2003/0162199 and Koukhareva et al., Nucl.Acids Symp. Ser. (Oxford), 259 (2008)). Agents that are used as hotstart mechanisms, such as antibodies, oligonucleotides, affibodies,chemical modifications, etc., may be referred to as “hot startinhibitors.”

In some embodiments, the hot start composition comprises an antibodyspecific for the polymerase. In some embodiments, the hot startcomposition comprises an antibody specific for the polymerase, which isbound to the polymerase. In some embodiments, the hot start compositioncomprises an inhibitor specific for the polymerase, which is bound tothe polymerase. In some embodiments, the inhibitor comprises anAffibody®. Affibodies are described, e.g., in U.S. Publication2012/0082981; see also Nord et al., 2000, J. Biotechnol. 80: 45-54; USPat. No. 6602977; Nygren, 2008, FEBS J. 275: 2668-2676; Nord et al.,1997, 15: 772-777; U.S. Patent No. 5831012. In some embodiments, theinhibitor comprises an oligonucleotide. In some embodiments, theinhibitor comprises a chemical modification.

As used herein, dual hot start reaction mixtures comprising “at leasttwo different mechanisms” encompass those reaction mixtures that maycomprise at least two different hot start mechanisms that functionsimilarly or use similar components. For example, dual hot startreaction mixtures can comprise reagents or reagent solutions designedfor two different antibody-based hot start mechanisms, or two differentoligonucleotide-based hot start mechanisms, or one antibody-based andone oligonucleotide-based hot start mechanism, or one antibody-based andone chemical modification-based hot start mechanism, or any suchcombination available.

Hot Start Antibodies and Methods of Making Same

In some embodiments, a hot start composition or dual hot startcomposition comprises an antibody inhibitor of a thermostable polymerasedescribed herein. In some embodiments, the antibody is a monoclonalantibody.

Methods for producing and screening for antibodies that are suitable foruse in hot start compositions with the polymerases described herein areknown in the art. In some embodiments, a hot start antibody inhibits thenucleic acid synthesis activity of the thermostable polymerase describedherein. In some embodiments, a hot start antibody inhibits exonucleaseactivity of the thermostable polymerase. In some embodiments, a hotstart antibody inhibits both the nucleic acid synthesis activity and theexonuclease activity of the thermostable polymerase.

In some embodiments, hot-start antibodies increase the specificity ofnucleic acid synthesis reactions, because they inactivate the polymeraseat room temperature, thus avoiding extension of nonspecificallyhybridized primers. The functional activity of the polymerase isrestored by disassociating the antibody from the polymerase, forexample, by incubating the composition at a higher temperature. In someembodiment, the “higher temperature” is from about 65° C. to about 99°C., from about 70° C. to about 99° C., 75° C. to about 99° C., or fromabout 80° C. to about 99° C., or from about 85° C. to about 99° C., orfrom about 90° C. to about 99° C., for a time period of at least 15seconds, or at least 30 seconds, or at least 1 minute, or at least 90seconds, or at least 2 minutes; to about 3 minutes, or about 4 minutes,or about 5 minutes, or about 7 minutes, or about 10 minutes, or more. Insome embodiments, the higher temperature is at least 60° C., at least65° C., at least 70° C., at least 75° C., at least 80° C., or at least85° C. In some embodiments, the temperature and duration of incubationto disassociate the antibody and activate the polymerase may bedetermined for the particular polymerase and antibody to be employed.

Methods for screening for antibodies of use in the present inventioninclude methods known in the art, such as affinity-based ELISA assays,as well as functional assays for polymerase and/or exonucleaseinhibition. For such functional assays, the amount of DNA produced ordigested per unit of time can be correlated to the activity of thepolymerase or exonuclease used, thus providing an estimate of the amountof inhibition a particular antibody can exert on either or both thepolymerase and exonuclease activity of the polymerase.

Antibodies may be produced using any method known in the art. As anonlimiting example, an antibody to a particular antigen (such as apolymerase described herein) may be produced by immunizing an animal(such as a mouse, rat, rabbit, goat, sheep, horse, etc.) with theantigen and isolating antibodies from the serum of the animal and/orimmortalizing primary B cells from the animal to produce hybridomas thatexpress the antibodies. Phage display technology may also be used toproduce antibodies that bind to the polymerases described herein. Phagedisplay libraries are commercially available and methods of selectingantibodies from such libraries are known in the art. See, e.g., Vaughanet al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998,Proc. Natl. Acad. Sci. (USA) 95:6157-6162; Hoogenboom and Winter, 1991,J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581.

Exemplary Assays to Determine Polymerase Processivity, Yield,Sensitivity, and Specificity

Polymerase processivity may be measured using various methods known inthe art. In some embodiments, processivity refers to the number ofnucleotides incorporated during a single binding event of polymerase toa primed template. As a nonlimiting example, a detectably labeled primermay be annealed to circular or linearized DNA to form a primed nucleicacid template. In measuring processivity, the primed nucleic acidtemplate may be present in significant molar excess to the polymerase toreduce the likelihood that any one primed template will be extended morethan once by a polymerase. A “significant molar excess” may be, forexample, a ratio of 500:1, or 1000:1, or 2000:1, or 4000:1, or 5000:1(primed DNA:DNA polymerase), etc., in the presence of suitable buffersand dNTPs. Nucleic acid synthesis may be initiated by adding, forexample, MgCl₂. Nucleic acid synthesis reactions are quenched at varioustimes after initiation, and analyzed by any appropriate method todetermine the length of the product. At a polymerase concentration wherethe median product length does not change with time or polymeraseconcentration, the length corresponds to the processivity of the enzyme.In some embodiments, the processivity of a polymerase described, such asa polymerase comprising a neutral amino acid at position correspondingto position 762 of SEQ ID NO: 1 may be compared to the processivity ofthe same polymerase without the neutral amino acid mutation.

In some embodiments, yield can be demonstrated by measuring the abilityof a polymerase to produce product. Increased yield can be demonstratedby determining the amount of product obtained in a reaction using apolymerase described herein (such as a polymerase comprising a neutralamino acid at position corresponding to position 762 of SEQ ID NO: 1),as compared to the amount of product obtained in a reaction carried outunder the same reaction conditions, but with the same polymerase withoutthe neutral amino acid mutation.

In some embodiments, long PCR may be used to determine enhancedprocessivity and yield. For example, an enzyme with enhancedprocessivity typically allows the amplification of a longer amplicons(>5 kb) in shorter extension times compared to an enzyme with relativelylower processivity.

Other methods of assessing efficiency of the polymerases of theinvention can be determined by those of ordinary skill in the art usingstandard assays of the enzymatic activity of a given modificationenzyme.

The sensitivity of a polymerase described herein may be determined bymeasuring the yield of nucleic acid synthesis product in a series ofreactions with differing copy numbers of nucleic acid template. In someembodiments, the template copy number at which a polymerase of theinvention (such as a polymerase comprising a neutral amino acid atposition corresponding to position 762 of SEQ ID NO: 1) producesdetectable product is compared to the template copy number at which thesame polymerase without the neutral amino acid mutation producesdetectable product. The lower the template copy number at which thepolymerase produces detectable product, the more sensitive thepolymerase.

In some embodiments, specificity of a polymerase may be measured bydetermining the ability of the polymerase to discriminate betweenmatched primer/template duplexes and mismatched primer/templateduplexes. In some embodiments, specificity is a measure of thedifference in the relative yield of two reactions, one of which employsa matched primer, and one of which employs a mismatched primer. In someembodiments, an enzyme with increased discrimination will have a higherrelative yield with the matched primer than with the mismatched primer.In some embodiments, a ratio of the yield with the matched primer versusthe mismatched primer is determined. In some embodiments, the ratio canbe compared to the yield obtained under the same reaction conditionsusing the parental polymerase.

DNA Synthesis Methods; Kits, Compositions, Systems, and Apparatuses

Provided herein are methods of synthesizing or amplifying DNA andrelated kits, compositions, systems, and apparatuses involving at leastone polymerase according to this disclosure. In some embodiments,reagents for nucleic acid synthesis are provided. In some embodiments,reagents for nucleic acid synthesis include any one or any combinationof target polynucleotides, particles attached with capture primers,solution-phase primers, fusion primers, other additional primers,enzymes (e.g., polymerases), accessory proteins (e.g., recombinase,recombinase loading protein, single-stranded binding protein, helicaseor topoisomerase), nucleotides, divalent cations, binding partners,co-factors and/or buffer. In some embodiments, reagents for nucleic acidsynthesis include a dUTPase as an accessory protein.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, methods, kits and apparatuses, comprisingone or more nucleotides. In some embodiments, the compositions (andrelated methods, systems, kits and apparatuses) includes one type, or amixture of different types of nucleotides. A nucleotide comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase. Suchnucleotides include not only naturally occurring nucleotides but alsoany analogs, regardless of their structure, that can bind selectivelyto, or can be polymerized by, a polymerase. While naturally occurringnucleotides typically comprise base, sugar and phosphate moieties, thenucleotides of the present disclosure can include compounds lacking anyone, some or all of such moieties. In some embodiments, the nucleotidecan optionally include a chain of phosphorus atoms comprising three,four, five, six, seven, eight, nine, ten or more phosphorus atoms. Insome embodiments, the phosphorus chain can be attached to any carbon ofa sugar ring, such as the 5′ carbon. The phosphorus chain can be linkedto the sugar with an intervening O or S. In some embodiments, one ormore phosphorus atoms in the chain can be part of a phosphate grouphaving P and O. In some embodiments, the phosphorus atoms in the chaincan be linked together with intervening O, NH, S, methylene, substitutedmethylene, ethylene, substituted ethylene, CNH2, C(O), C(CH2), CH2CH2,or C(OH)CH2R (where R can be a 4-pyridine or 1-imidazole). In someembodiments, the phosphorus atoms in the chain can have side groupshaving O, BH3, or S. In the phosphorus chain, a phosphorus atom with aside group other than O can be a substituted phosphate group. In thephosphorus chain, phosphorus atoms with an intervening atom other than Ocan be a substituted phosphate group. Some examples of nucleotideanalogs are described in Xu, U.S. Pat. No. 7,405,281.

Some examples of nucleotides that can be used in the disclosedcompositions (and related methods, systems, kits and apparatuses)include, but are not limited to, ribonucleotides, deoxyribonucleotides,modified ribonucleotides, modified deoxyribonucleotides, ribonucleotidepolyphosphates, deoxyribonucleotide polyphosphates, modifiedribonucleotide polyphosphates, modified deoxyribonucleotidepolyphosphates, peptide nucleotides, modified peptide nucleotides,metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano- moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof. In some embodiments, a nucleotide can include apurine or pyrimidine base, including adenine, guanine, cytosine,thymine, uracil or inosine. In some embodiments, a nucleotide includesdATP, dGTP, dCTP, dTTP and dUTP.

In some embodiments, the nucleotide is unlabeled. In some embodiments,the nucleotide comprises a label and referred to herein as a “labelednucleotide”. In some embodiments, the label can be in the form of afluorescent dye attached to any portion of a nucleotide including abase, sugar or any intervening phosphate group or a terminal phosphategroup, i.e., the phosphate group most distal from the sugar.

In some embodiments, the disclosure relates generally to compositions,as well as related systems, methods, kits and apparatuses, comprisingany one or any combination of capture primers, reverse solution-phaseprimers, fusion primers, target polynucleotides and/or nucleotides thatare non-labeled or attached to at least one label. In some embodiments,the label comprises a detectable moiety. In some embodiments, the labelcan generate, or cause to generate, a detectable signal. In someembodiments, the detectable signal can be generated from a chemical orphysical change (e.g., heat, light, electrical, pH, salt concentration,enzymatic activity, or proximity events). For example, a proximity eventcan include two reporter moieties approaching each other, or associatingwith each other, or binding each other. In some embodiments, thedetectable signal can be detected optically, electrically, chemically,enzymatically, thermally, or via mass spectroscopy or Ramanspectroscopy. In some embodiments, the label can include compounds thatare luminescent, photoluminescent, electroluminescent, bioluminescent,chemiluminescent, fluorescent, phosphorescent or electrochemical. Insome embodiments, the label can include compounds that are fluorophores,chromophores, radioisotopes, haptens, affinity tags, atoms or enzymes.In some embodiments, the label comprises a moiety not typically presentin naturally occurring nucleotides. For example, the label can includefluorescent, luminescent or radioactive moieties.

In some embodiments, the nucleic acid synthesis reaction includes acycled amplification reaction, such as a polymerase chain reaction (PCR)(U.S. Pat. 4,683,195 and 4,683,202 both granted to Mullis). Multipleexamples of PCR according to this disclosure are provided below. In someembodiments, the nucleic acid synthesis reaction includes an isothermalreaction, such as an isothermal self-sustained sequence reaction (Kwoh1989 Proceedings National Academy of Science USA 86:1173-1177; WO1988/10315; and U.S. patents 5,409,818, 5,399,491, and 5,194,370), or arecombinase polymerase amplification (RPA) (U.S. Pat. No. 5,223,414 toZarling, U.S. Pat. Nos. 5,273,881 and 5,670,316 both to Sena, and U.S.Pat. Nos. 7,270,981, 7,399,590, 7,435,561, 7,666,598, 7,763,427,8,017,339, 8,030,000, 8,062,850, and 8,071,308).

PCR is a nucleic acid synthesis reaction in which the reaction mixtureis subjected to reaction cycles, each reaction cycle comprising adenaturation period and at least one annealing and/or extension period,resulting if successful in synthesis of copies of a nucleic acidtemplate in at least the initial cycles, and copies of the copies in atleast the later cycles, generally resulting in exponential amplificationof the template. In PCR, in some instances, a pair of primers areprovided that bind at each end of a target region, on opposite strandssuch that they each prime synthesis toward the other primer. Thereaction is thermocycled so as to drive denaturation of the substrate ina high temperature step, annealing of the primers at a lower temperaturestep, and extension at a temperature which may be but is not necessarilyhigher than that of the annealing step. Exponential amplification occursbecause the products of one cycle can serve as template in the nextcycle.

An embodiment of isothermal self-sustained sequence reaction, alsosometimes referred to as transcription-mediated amplification or TMA,involves synthesizing single-stranded RNA, single-stranded DNA anddouble-stranded DNA. The single-stranded RNA is a first template for afirst primer, the single-stranded DNA is a second template for a secondprimer, and the double stranded DNA is a third template for synthesis ofa plurality of copies of the first template. A sequence of the firstprimer or the second primer is complementary to a sequence of a targetnucleic acid and a sequence of the first primer or the second primer ishomologous to a sequence of the target nucleic acid. In an embodiment ofan isothermal self-sustained sequence reaction, a first cDNA strand issynthesized by extension of the first primer along the target by anenzyme with RNA-dependent DNA polymerase activity, such as a reversetranscriptase. The first primer can comprise a polymerase bindingsequence (PBS) such as a PBS for a DNA-dependent RNA polymerase, such asT7, T3, or SP6 RNA polymerase. The first primer comprising a PBS issometimes referred to as a promoter-primer. The first cDNA strand isrendered single-stranded, such as by denaturation or by degradation ofthe RNA, such as by an RNase H. The second primer then anneals to thefirst cDNA strand and is extended to form a second cDNA strand by a DNApolymerase activity. Forming the second cDNA strand renders the cDNAdouble-stranded, including the PBS. RNA can then be synthesized from thecDNA, which comprises the PBS, by a DNA-dependent RNA polymerase, suchas T7, T3, or SP6 RNA polymerase, thereby providing a template forfurther events (extension of the first primer, rendering the productsingle-stranded, extension of the second primer, and RNA synthesis).Exponential amplification occurs because the RNA product cansubsequently serve as a template and also because RNA products can begenerated repeatedly from a cDNA comprising the PBS.

An embodiment of RPA can be performed isothermally and employs arecombinase to promote strand invasion of a double-stranded template byforward and reverse primers. The 3′ ends of the primers are extended,displacing template strands at least in part. Subsequent strandinvasion/annealing events, including to previously produced extensionproducts, occur and are followed by extension, resulting inamplification. In some embodiments, recombinase activity is supported bythe presence of one or more recombinase accessory proteins, such as arecombinase loading protein and/or single-stranded binding protein.

In some embodiments, the disclosure relates generally to compositions,and related methods, systems, kits and apparatuses, comprising a nucleicacid synthesis reaction (synthesis condition) that can be conductedunder thermocycling or isothermal conditions, or a combination of bothtypes of conditions. For example, the synthesis condition can includealternating between thermocycling and isothermal synthesis conditions,in any order.

In some embodiments thermocycling synthesis conditions comprise anucleic acid synthesis reaction mixture that is subjected to an elevatedtemperature for a period of time that is sufficient to denature at leastabout 30-95% of the double-stranded target nucleic acids, and thensubjected to a lower temperature for a period of time that is sufficientto permit hybridization between the single-stranded target nucleic acidsand any of the primers (e.g., capture primer, reverse solution-phaseprimer, or fusion primer). In some embodiments, the increase anddecrease temperature cycle is repeated at least once.

In some embodiments isothermal synthesis conditions comprise a nucleicacid synthesis reaction mixture that is subjected to a temperaturevariation which is constrained within a limited range during at leastsome portion of the synthesis, including for example a temperaturevariation is within about 20° C., or about 10° C., or about 5° C., orabout 1-5° C., or about 0.1-1° C., or less than about 0.1° C.

In some embodiments, an isothermal nucleic acid synthesis reaction canbe conducted for about 2, 5, 10, 15, 20, 30, 40, 50, 60 or 120 minutes,or longer. In some embodiments, an isothermal nucleic acid synthesisreaction can be conducted for at least about 2 minutes. In someembodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 120 minutes or less. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 2to about 120 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 2 to about 60 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 60 to about 120 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 2to about 5 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 5 to about 10 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 10 to about 15 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 10to about 15 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 10 to about 15 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 15 to about 20 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 20to about 30 minutes. In some embodiments, an isothermal nucleic acidsynthesis reaction can be conducted for about 30 to about 40 minutes. Insome embodiments, an isothermal nucleic acid synthesis reaction can beconducted for about 40 to about 50 minutes. In some embodiments, anisothermal nucleic acid synthesis reaction can be conducted for about 50to about 60 minutes.

In some embodiments, an isothermal nucleic acid synthesis reaction canbe conducted at about 15-30° C., or about 30-45° C., or about 45-60° C.,or about 60-75° C., or about 75-90° C., or about 90-93° C., or about93-99° C.

In some embodiments, the disclosure relates generally to methods, andrelated compositions, systems, kits and apparatuses, that furtherinclude an enrichment step. In some embodiments, an enrichment stepcomprises a pre-amplification reaction. See, e.g., U.S. Pat. No.8,815,546 B2 . As a nonlimiting example, a pre-amplification reactionmay comprise random primers to amplify a portion, even a substantialportion, of the nucleic acid template in a sample. In this manner, theoverall amount of nucleic acid template may be increased prior to asequence-specific nucleic acid synthesis reaction.

In some embodiments, an amplified population of nucleic acids caninclude an affinity moiety. For example, in conducting any of thenucleic acid synthesis methods according to the present teachings, asolution-phase/reverse primer that is attached to an affinity moiety(e.g., biotin) can be used to conduct a synthesis reaction to produce anamplified population of nucleic acids that are attached to the affinitymoiety. In some embodiments, the enrichment step comprises forming aenrichment complex by binding the affinity moiety (which is attached tothe amplified population of nucleic acids) with a purification particle(e.g., paramagnetic bead) that is attached to a receptor moiety (e.g.,streptavidin). An example of purification particles include MyOne™ Beadsfrom Dynabeads, which are paramagnetic beads attached to streptavidin.In some embodiments, a magnet can be used to separate/remove theenrichment complex from amplified population of nucleic acids that lackthe affinity moiety. In some embodiments, the enrichment step can berepeated at least once. In some embodiment, the enrichment step isfollowed by one or more washing step.

In some embodiments, the disclosure relates generally to methods, andrelated compositions, systems, kits and apparatuses that further includeat least one washing step. The washing step can be conducted at any timeduring the workflow for nucleic acid synthesis. In some embodiments, awashing step can remove excess or unreacted components of the nucleicacid synthesis or enrichment reactions.

In some embodiments, any of the nucleic acid synthesis methods, orenrichment steps, according to the present teachings, can be conductedmanually or by automation. In some embodiments, any one or anycombination of the steps can be conducted manually or by automation,including: conducting a nucleic acid synthesis reaction, enriching,and/or washing. For example, any reagents for a nucleic acid synthesis,enrichment or washing, can be deposited into, or removed from, areaction vessel via manual or automated modes.

In various embodiments, the disclosure relates to compositionscomprising at least one polymerase described herein. In someembodiments, the composition is a hot start composition. In some suchembodiments, the composition is a dual hot start composition. In someembodiments, the dual hot start composition comprises at least twodifferent hot start mechanisms that are used to inhibit or substantiallyinhibit the polymerase activity at a first temperature. Such hot startmechanisms include, but are not limited to, antibodies or combinationsof antibodies that block DNA polymerase activity at lower temperatures,antibody mimetics or combinations of antibody mimetics that block DNApolymerase activity at lower temperatures (such as, for example,Affibodies®), oligonucleotides that block DNA polymerase activity atlower temperatures (such as, for example, aptamers), reversible chemicalmodifications of the DNA polymerase that dissociate at elevatedtemperatures, amino acid modifications of the DNA polymerase thatprovide reduced activity at lower temperatures, fusion proteins thatinclude hyperstable DNA binding domains and topoisomerase, othertemperature dependent ligands that inhibit the DNA polymerase, singlestranded binding proteins that sequester primers at lower temperatures,modified primers or modified dNTPs. Hot start compositions, in someembodiments, comprise at least one polymerase described herein with orwithout a hot start chemical modification, at least one hot startantibody, at least one hot start aptamer, and/or at least one hot startAffibody®. In some embodiments, a hot start composition comprises atleast one polymerase described herein with or without a hot startchemical modification, at least one hot start antibody and at least onehot start aptamer or at least one hot start Affibody®. In someembodiments, a hot start composition comprises at least one polymerasedescribed herein with or without a hot start chemical modification, atleast one hot start Affibody® and at least one hot start antibody or atleast one hot start aptamer. In some embodiments, a hot startcomposition comprises a polymerase described herein with or without ahot start chemical modification, a hot start antibody, and a hot startaptamer or a hot start Affibody®. In some embodiments, a hot startcomposition comprises a polymerase described herein with or without ahot start chemical modification, a hot start Affibody®, and a hot startantibody or a hot start aptamer. In some embodiments, a hot startcomposition comprises a polymerase described herein with or without ahot start chemical modification, a hot start antibody, and a hot startAffibody®. In some embodiments, a hot start composition comprises apolymerase described herein with or without a hot start chemicalmodification, a hot start antibody, and a hot start aptamer.

In some embodiments, a composition comprises one or more detergents, oneor more protein stabilizers, and/or at least one UTPase. In someembodiments, a composition comprises one or more detergents, one or moreprotein stabilizers, and at least one UTPase. In some embodiments, acomposition comprises at least one monovalent cationic salt, at leastone divalent cationic salt, and/or at least one dNTP. In someembodiments, a composition further comprises at least one dye. In someembodiments, a composition comprises additional stabilizers thatincrease the density of the composition.

Nonlimiting exemplary detergents that may be used in the compositionsprovided herein include nonionic, ionic (anionic, cationic) andzwitterionic detergents. Exemplary such detergents include, but are notlimited to, Hecameg(6-O-(N-Heptylcarbamoyl)-methyl-α-D-glucopyranoside), Trition X-200,Brij-58, CHAPS, n-Dodecyl-b-D-maltoside, NP-40, sodium dodecyl sulphate(SDS), TRITON® X-15, TRITON® X-35, TRITON® X-45, TRITON® X-100, TRITON®X-102, TRITON® X-114, TRITON® X-165, TRITON® X-305, TRITON® X-405,TRITON® X-705, Tween® 20 and/or ZWITTERGENT®. Other detergents may alsobe suitable, as may be determined by one of skill in the art. See, e.g.,U.S. Pat. No. 7972828B2, U.S. Pat. No. 8980333B2 U.S. Publication No.2008/0145910; U.S. Publication No. 2008/0064071; U.S. Pat. No.6,242,235; U.S. Pat. No. 5,871,975; and U.S. Pat. No. 6,127,155 forexemplary detergents.

Nonlimiting exemplary protein stabilizers that may be used in thecompositions provided herein include BSA, inactive polymerases (such asinactivated Taq polymerase; see, e.g., US Publication No. 2011/0059490),and apotransferrin. Further nonlimiting exemplary stabilizers that maybe used in the compositions provided herein include glycerol, trehalose,lactose, maltose, galactose, glucose, sucrose, dimethyl sulfoxide(DMSO), polyethylene glycol, and sorbitol.

Nonlimiting exemplary UTPases that may be used in the compositionsprovided herein include UTPases from thermophilic bacteria. See, e.g.,PNAS, 2002, 99: 596-601.

Nonlimiting exemplary dyes that may be used in the compositions providedherein include xylene cyanol FF, tartrazine, phenol red, quinolineyellow, zylene cyanol, Brilliant Blue, Patent Blue, indigocarmine, acidred 1, m-cresol purple, cresol red, neutral red, bromocresol green, acidviolet 5, bromo phenol blue, and orange G (see, e.g., US Pat. No.8663925 B2). Additional nonlimiting exemplary dyes are described, e.g.,in US Pat. No. 6,942,964. One skilled in the art will appreciate thatany dye that does not inhibit nucleic acid synthesis by the polymerasesdescribed herein may be used.

In some embodiments, a storage composition is provided comprising apolymerase provided herein, at least one hot start antibody, at leastone protein stabilizer, and at least one UTPase, in a buffer suitablefor storage. In some embodiments, a storage composition is providedcomprising a polymerase provided herein, at least one hot startantibody, at least one Affibody®, at least one protein stabilizer, andat least one UTPase, in a buffer suitable for storage. In someembodiments, a storage composition is provided comprising a polymeraseprovided herein, two hot start antibodies, a protein stabilizer, and aUTPase, in a buffer suitable for storage. In some embodiments, thestorage buffer comprises a buffering agent (such as Tris HCl), a salt(such as KCl or NaCl), a stabilizer (such as glycerol), a reducing agent(such as DTT), a divalent cation chelating agent (such as EDTA), and adetergent (such as hecameg and/or Triton X-200 and/or NP-40 and/orTween-20, etc.). In some embodiments, the storage composition comprises0.5 to 5 units (U), or 0.5 to 3U, or 1 to 3U, or 2U of polymerase perµl. In some embodiments, the storage composition comprises 0.05 to 1mg/ml, or 0.05 to 0.5 mg/ml, or 0.1 to 0.5 mg/ml, or 0.1 to 0.3 mg/ml ofeach hot start antibody. In some embodiments, the storage compositioncomprises 0.1 to 10 mg/ml, or 0.1 to 5 mg/ml, or 0.5 to 5 mg/ml, or 0.5to 2 mg/ml of each hot start Affibody®. In some embodiments, the storagecomposition comprises 0.5 to 5 mg/ml, or 1 to 5 mg/ml, or 1 to 3 mg/mlof each protein stabilizer.

In some embodiments, a reaction composition is provided, comprising atleast one polymerase described herein, at least one buffering agent(such as Tris HCl), at least one monovalent cationic salt (such as KClor NaCl), at least one divalent cationic salt (such as MgCl₂ or MnCl₂),at least one detergent (such as hecameg and/or Triton X-200 and/or NP-40and/or Tween-20, etc.), and at least one dNTP. In some embodiments, thecomposition comprises dATP, dCTP, dGTP, and dTTP. In some embodiments,the reaction composition further comprises at least one dye. In someembodiments, for example when the composition is to be loaded on a gel,the reaction composition comprises additional stabilizers that increasethe density of the composition, such as polyethylene glycol (e.g., PEG4000) and/or sucrose. PEG 4000 may be included, in some embodiments, ata concentration of 0.5-2%, or about 1%; and sucrose may be included, insome embodiments, at a concentration of 1-5%, or 1-3%, or about 2% (or2-10%, or 2-6%, or about 4% for a 2X reaction composition). In someembodiments, the buffering agent (such as Tris HCl) is present at aconcentration of 5-50 mM, or 5-30 mM, or 5-20 mM (or 10-100 mM, or 10-60mM, or 10-40 mM for a 2X reaction composition). In some embodiments, themonovalent cation (such as K+ or Na+) is present at a concentration of50-300 mM, or 50-200 mM, or 75-150 mM, or about 110 mM (or 100-600 mM,or 100-400 mM, or 150-300 mM, or about 220 mM for a 2X reactioncomposition). In some embodiments, a detergent (such as hecameg and/orTriton X-200 and/or NP-40 and/or Tween-20, etc.) is present at aconcentration of 0.05-0.3%, or 0.1-0.2%, or about 0.15% (or 0.01-0.6%,or 0.2-0.4%, or about 0.3% for a 2X reaction composition). In someembodiments, the Mg²⁺ or Mn²⁺ is present at a concentration of 0.5-5 mM,or 0.5-3 mM, or about 1.5 mM (or 1-10 mM, or 1-6 mM, or about 3 mM for a2X reaction composition). In some embodiments, each dNTP is present at aconcentration of 0.05-1 mM, or 0.1-0.8 mM, or 0.1-0.6 mM, or 0.1-0.4 mM,or about 0.2 mM (or 0.1-2 mM, or 0.2-1.6 mM, or 0.2-1.2 mM, or 0.2-0.8mM, or about 0.4 mM for a 2X reaction composition).

PCR enhancing factors may also be used to improve efficiency of theamplification. As used herein, a “PCR enhancing factor” or a “PolymeraseEnhancing Factor” (PEF) refers to a complex or protein possessingpolynucleotide polymerase enhancing activity (Hogrefe et al., 1997,Strategies 10:93-96; and U.S. Pat. No. 6,183,997, both of which arehereby incorporated by references). For Pfu DNA polymerase, for example,PEF may comprise either P45 in native form (as a complex of P50 and P45)or as a recombinant protein. In the native complex of Pfu P50 and P45,only P45 exhibits PCR enhancing activity. The P50 protein is similar instructure to a bacterial flavoprotein. The P45 protein is similar instructure to dCTP deaminase and dUTPase, but it functions only as adUTPase converting dUTP to dUMP and pyrophosphate. PEF, according to thepresent disclosure, may also be selected from the group consisting of:an isolated or purified naturally occurring polymerase enhancing proteinobtained from an archaeabacteria source (e.g., Pyrococcus furiosus); awholly or partially synthetic protein having the same amino acidsequence as Pfu P45, or analogs thereof possessing polymerase enhancingactivity; polymerase-enhancing mixtures of one or more of said naturallyoccurring or wholly or partially synthetic proteins;polymerase-enhancing protein complexes of one or more of said naturallyoccurring or wholly or partially synthetic proteins; orpolymerase-enhancing partially purified cell extracts containing one ormore of said naturally occurring proteins (U.S. Pat. No. 6,183,997,supra).

In some embodiments, a reaction composition further comprisesingredients that enhance nucleic acid synthesis from high GC-contenttemplates. In some such embodiments, the reaction composition comprisesglycerol, DMSO, and/or ammonium sulphate. In some embodiments, thereaction composition comprises glycerol, DMSO, and ammonium sulphate. Insome embodiments, glycerol is present in the reaction composition at aconcentration of 5-20%, or 5-15%, or about 10%. In some embodiments,DMSO is present in the reaction composition at a concentration of 1-10%,or 3-10%, or 3-7%, or about 5%. In some embodiments, ammonium sulphateis present in the reaction composition at 10-50 mM, or 15-40 mM, or20-30 mM, or about 25 mM.

In some embodiments, a reaction composition is provided at 2X, 5X, 10X,etc. concentration, in which case, the concentrations discussed hereinare multiplied (e.g., as noted above; doubled for 2X). A 2X reactioncomposition is typically diluted by 2-fold, for example, when thetemplate nucleic acid and/or primers are added to the composition.

In some embodiments, a reaction composition comprises nucleic acidtemplate and at least one primer for nucleic acid synthesis. In someembodiments, each primer is included in the reaction composition at aconcentration of 0.1-0.8 µM, or 0.1-0.5 µM, or 0.2-0.4 µM, or about 0.3µM. One skilled in the art will appreciate that the template nucleicacid may be provided at a wide range of concentrations, which lowerlimit, in some embodiments, may be determined by the sensitivity of thepolymerase.

In some embodiments, the composition comprises at least one PCRinhibitor. In some embodiments, the PCR inhibitor comprises xylan,heparin, humic acid, or SDS. In some embodiments, methods according tothe disclosure comprise amplifying DNA in the presence of at least onePCR inhibitor. In some embodiments, the PCR inhibitor comprises xylan.In some embodiments, the PCR inhibitor comprises heparin.

In various embodiments, the composition may be an aqueous composition.In various embodiments, the composition may be a lyophilizedcomposition. In some embodiments, the composition comprises acryoprotectant and/or a preservative and/or other additives known tothose skilled in the art. Nonlimiting exemplary cryoprotectants andpreservatives include, for example, the stabilizers and reducing agentsdescribed herein.

Nucleic Acids; Vectors; Host Cells; Methods of Production and/orPurification

Provided herein are nucleic acids comprising a sequence encoding apolymerase according to this disclosure. In some embodiments, thenucleic acid is operably linked to a promoter. In some embodiments, thepromoter is a promoter for a bacteriophage RNA polymerase, such as a T7promoter. In some embodiments, the nucleic acid is codon-optimized forexpression in a host cell, such as a microorganism, e.g., a bacterium,such as E. coli.

Also provided herein are vectors comprising any of the nucleic acidscomprising a sequence encoding a polymerase according to this disclosurediscussed above. In some embodiments, the vector is a plasmid. In someembodiments, the vector is an expression vector. In some embodiments,the vector contains a selectable marker. In some embodiments, the vectoris capable of being propagated in a microorganism, e.g., a bacterium,such as E. coli.

Also provided herein are host cells comprising any of the nucleic acidscomprising a sequence encoding a polymerase according to this disclosurediscussed above. Also provided herein are host cells comprising any ofthe vectors comprising a sequence encoding a polymerase according tothis disclosure discussed above. In some embodiments, the host cell is amicroorganism, e.g., a bacterium, such as E. coli. In some embodiments,the host cell further comprises a nucleic acid encoding a heterologousRNA polymerase. In some embodiments, the heterologous RNA polymerase isa bacteriophage RNA polymerase, such as bacteriophage T7 RNA polymerase.In some embodiments, the heterologous RNA polymerase is operably linkedto a promoter, such as an inducible promoter, e.g., a lac-induciblepromoter. In some embodiments, the host cell is of a protease-deficientstrain. In some embodiments, the host cell is E. coli BL-21. In someembodiments, the host cell, such as BL-21, is modified to carry tRNAgenes encoding tRNAs with rarer anticodons (for example, argU, ileY,leuW, and proL tRNA genes).

Also provided herein are methods of producing and/or purifying apolymerase according to this disclosure. In some embodiments, such amethod comprises culturing at least one host cell comprising a nucleicacid encoding a thermophilic DNA polymerase according to thisdisclosure, wherein the at least one host cell expresses thethermophilic DNA polymerase. In some embodiments, such a methodcomprises isolating a polymerase according to this disclosure from hostcells that have expressed the polymerase. In some embodiments, theisolating comprises lysing the host cells. In some embodiments, theisolating comprises heat treatment to denature host proteins. In someembodiments, denatured host proteins are removed, e.g., bycentrifugation. In some embodiments, the polymerase is purified viachromatography. Examples of procedures for purifying DNA polymerases areprovided, e.g., in Lawyer et al. (1993, PCR Meth. & App. 2: 275)(designed originally for the isolation of Taq polymerase) and Kong etal. (1993, J. Biol. Chem. 268: 1965) (involving a heat denaturation stepof host proteins, and two column purification steps over DEAE-Sepharoseand heparin-Sepharose columns).

EXAMPLES

The following examples are provided to illustrate certain disclosedembodiments and are not to be construed as limiting the scope of thisdisclosure in any way.

Example 1. Tolerance of Inhibitors by a Thermophilic DNA PolymeraseAccording to the Disclosure

The inhibitor resistance of a thermophilic DNA polymerase with thesequence of SEQ ID NO: 20 (including a Q at position 762) (762Qpolymerase) was compared to a version with a K at position 762 (762Kpolymerase) by amplifying PCR fragments in the presence of variousamounts of polymerase inhibitors. The performance of a thermophilic DNApolymerase with the sequence of SEQ ID NO: 22 (including a Q at position762 and S at position 408) (408S 762Q polymerase) was compared to aversion with a K at position 762 (408S 762K polymerase) by amplifyingPCR fragments in the presence of various amounts of polymeraseinhibitors.

Heparin. A 2 kb fragment was amplified from 20 ng of human genomic DNAtemplate in 20 µl PCR reactions in the presence of 0 to 0.3 µM ofheparin using the thermophilic DNA polymerases (FIG. 1 ). Primers withthe following sequences were used:

-   GAAGAGCCAAGGACAGGTAC (SEQ ID NO: 64) (forward);-   CCTCCAAATCAAGCCTCTAC (SEQ ID NO: 65) (reverse). The PCR program was    as follows:

98° C. 30 s 98° C. 10 s 60° C. 10 s x30 72° C. 1 min 72° C. 5 min

Products were detected by agarose gel electrophoresis and staining withEthidium bromide. Detectable product was observed at up to 0.25 µMheparin for the 762Q polymerase and 0.15 µM for 408S 762Q polymerase.Products were not detected at or above 0.1 µM heparin for the 762K and408S 762K polymerases.

Xylan. A 2 kb fragment was amplified from 40 ng of human genomic DNAtemplate in 20 µl PCR reactions in the presence of 0 to 400 ng/µl xylanusing the thermophilic DNA polymerases (FIG. 2 ). The primers, PCRprogram, and product detection were as described above with respect toheparin.

Detectable product was observed at up to 400 ng/µl xylan for the 762Qpolymerase. Products were not detected at 400 ng/µl xylan for the 762Kpolymerases.

Humic acid. A 2 kb fragment was amplified from 40 ng of human genomicDNA template in 20 µl PCR mixture in the presence of 0 to 1.0 ng/ml ofhumic acid using a thermophilic DNA polymerase with the sequence of SEQID NO: 20 (including a Q at position 762) was compared to a version witha K at position 762 (FIG. 3 ). Primers, the PCR program, and productdetection were as described above with respect to heparin.

Sodium dodecyl sulfate. A 2 kb fragment was amplified from 40 ng ofhuman genomic DNA template in 20 µl PCR mixture in the presence of 0 to0.016% or 0.2% (w/v) sodium dodecyl sulfate (SDS) using the 762Q or 762Kpolymerases (FIG. 4 ). The primers, PCR program, and product detectionwere as described above with respect to heparin.

Thus, increased inhibitor tolerance was observed for the polymeraseswith a Q at position 762.

Example 2. Yield and Sensitivity of PCR With a Thermophilic DNAPolymerase According to the Disclosure

The PCR performance (sensitivity and yield) of the 762Q and 762Kpolymerases discussed in Example 1 were compared by amplifying PCRfragments from various amounts of DNA template.

Sensitivity. A 2 kb fragment was amplified from a series of amounts ofhuman genomic DNA template between 0 and 400 ng in a 20 µl PCR mixtureusing the thermophilic DNA polymerases (FIG. 5 ). The primers and thePCR program were the same as those used in Example 1. Products wereanalyzed by agarose gel electrophoresis and stained as in Example 1.

The results showed that the reaction with the 762Q polymerase had highersensitivity (amplification from lower amounts of template DNA) than withthe 762K polymerase, and the reaction with the 408S 762Q polymerase hadhigher sensitivity than with the 408S 762K polymerase.

Yield. A 10 kb fragment was amplified from a series of amounts of phagelambda DNA template between 0 and 200 ng in a 20 µl PCR mixture usingthe thermophilic DNA polymerases. The primers were:CAGTGCAGTGCTTGATAACAGG (SEQ ID NO: 66) (forward) andGTAGTGCGCGTTTGATTTCC (SEQ ID NO: 67) (reverse). The PCR program was:

98° C. 30 s 98° C. 10 s 60° C. 15 s x25 72° C. 150 s 72° C. 10 min

Products were analyzed by agarose gel electrophoresis and stained as inExample 1. The results from two experiments showed that the reactionsensitivity (amplification from lower amounts of template DNA) wassimilar for all the polymerases (FIG. 6A), while the 762Q polymeraseshowed the highest yield (140% from the 762K polymerase) amplifying the10 kb fragment from 0.5 ng of the template (FIG. 6B).

Example 3 Fidelity of Thermophilic DNA Polymerases According to theDisclosure

Polymerase fidelity was measured by next generation sequencing.Fragmented E.coli DNA (~300 bp) was amplified by Taq polymerase, 762Kpolymerase, 762Q polymerase, 408S 762K polymerase and 408S 762Qpolymerase. The number of effective PCR cycles was found by qPCR. Theamplified libraries were subjected to paired-end Illumina sequencingtogether with control E.coli PCR-free libraries. The polymerase errorrates were calculated using bioinformatics techniques. The backgroundlevel of experimental errors was estimated from PCR-free librarysequencing data. The polymerase introduced errors were identified asnucleotide changes in both pair-end reads, while nucleotide changes inonly pair-end one read have been treated as instrumental errors and wereeliminated. The polymerase fidelities (1/error rate) were normalized tothe fidelity of Taq polymerase, which fidelity value is indicated as 1x.

The fidelity of the 762K polymerase was ~50X of the Taq polymerase, the762Q polymerase also showed similar fidelity (Table 1). The error ratesfor the 408S 762K and A408S 762Q polymerases were almostindistinguishable from the background, which indicate >100X fidelity ofthe Taq polymerase and is the threshold of fidelity measurements usingthis particular experimental setup. A thermophilic DNA polymerase withthe sequence of SEQ ID NO: 95 (including a H at position 36, S at aposition 408 and Q at position 762; “36H 408S 762Q polymerase”) was alsofound to have a fidelity of > 100 x of the Taq polymerase.

TABLE 1 Polymerase Fidelity, xTaq polymerase fidelity Taq 1 × 762K 20-70× 762Q 20-70 × 408S 762K >100 × * 408S 762Q >100 × * * - 100 x Taqfidelity is the threshold of fidelity measurements

Example 4. Yield and Sensitivity of PCR With a Thermophilic DNAPolymerase According to the Disclosure Provided as Hot-StartCompositions

The performance of a thermophilic DNA polymerase with the sequence ofSEQ ID NO: 22 (including a Q at position 762 and S at position 408)(408S 762Q polymerase) was compared to a version with a K at position762 (408S 762K polymerase) by amplifying various PCR fragments, with thepolymerases being provided as dual hot-start compositions. A dUTPase wasalso supplied in the reactions.

2 kb fragment from human genomic DNA template. The template was humangenomic DNA in a series of amounts between 0 and 400 ng in 20 µlreactions. The primers and the PCR program were the same as in Example1.

The 408S 762Q polymerase showed increased yield and higher sensitivityrelative to the 408S (FIG. 7A).

5 kb fragment from phage lambda DNA template. PCR primers wereCCTGCTCTGCCGCTTCACGC (SEQ ID NO: 68) (forward) andCGAACGTCGCGCAGAGAAACAGG (SEQ ID NO: 69) (reverse). The PCR program was:

98° C. 30 s 98° C. 10 s x30 72° C. 1 min 40 s 72° C. 10 min

Lambda DNA was provided as template at amounts between 0 and 200 ng in20 µl reactions. Products were analyzed by agarose gel electrophoresisand stained as in Example 1. Sensitivity was higher for reactions withthe 408S 762Q polymerase (FIG. 7B).

20 kb fragment from phage lambda DNA template. PCR primers wereCTGATGAGTTCGTGTCCGTACAACTGGCGTAATC (SEQ ID NO: 70) (forward) andGTGCACCATGCAACATGAATAACAGTGGGTTATC (SEQ ID NO: 71) (reverse). The PCRprogram was:

98° C. 30 s 98° C. 10 s x25 72° C. 10 min 72° C. 10 min

Lambda DNA was provided as template at amounts between 0 and 100 ng in20 µl reactions. Products were analyzed by agarose gel electrophoresisand stained as in Example 1. Band intensities from lower amounts of thetemplate were generally greater for reactions with the 408S 762Qpolymerase, indicating increased yield and sensitivity (FIG. 8 ).

20 kb fragment from Escherichia coli genomic DNA template. PCR primerswere:

-   GGGCGTTTTCCGTAACACTG (SEQ ID NO: 72) (forward) and-   TGACCACATACAATCGCCGT (SEQ ID NO: 73) (reverse). The PCR program was:

98° C. 30 s 98° C. 10 s 61° C. 30 s x30 72° C. 10 min 72° C. 10 min

E. coli gDNA template was provided as template at amounts between 0 and40 ng in 20 µl reactions. Products were analyzed by agarose gelelectrophoresis and stained as in Example 1.

Band intensities from lower amounts of the template were generallygreater for reactions with the 408S 762Q polymerase, indicatingincreased yield and sensitivity (FIG. 9 ).

7.5 kb fragment from human genomic DNA template. The template was humangenomic DNA in a series of amounts between 0 and 400 ng in 20 µlreactions. Primers were:

-   CTCCACAGGGTGAGGTCTAAGTGATGACA (SEQ ID NO: 74) (forward) and-   CAATCTCAGGGCAAGTTAAGGGAATAGTG (SEQ ID NO: 75) (reverse). The PCR    program was:

98° C. 30 s x30 98° C. 10 s 72° C. 180 s 72° C. 10 min

Band intensities were greater for reactions with the 408S 762Qpolymerase, indicating increased yield and sensitivity (FIG. 10 ).

Example 5 Fidelity of Thermophilic DNA Polymerases Provided as Hot-StartCompositions

Polymerase fidelity was measured by next generation sequencing.Fragmented E.coli DNA (~300 bp) was amplified by Taq polymerase, 408Spolymerase, and 408S 762Q polymerase with the polymerases being providedas dual hot-start compositions, including affibodies and antibodies. AdUTPase was also supplied in the reactions. Polymerase fidelities weremeasured as in the Example 3.

The error rates for the 408S and 408S 762Q polymerases were almostindistinguishable from the background, which indicate > 100X fidelity ofthe Taq polymerase and is the threshold of fidelity measurements usingthis particular experimental setup (Table 2).

TABLE 2 Polymerase Fidelity, xTaq polymerase fidelity Taq 1 × 408S >100× * 408S 762Q >100 × * * - 100 x Taq fidelity is the threshold offidelity measurements

Example 6. Tolerance of dUTP by a Thermophilic DNA Polymerase Accordingto the Disclosure

Tolerance of dUTP as a replacement for dTTP of a thermophilic DNApolymerase with the sequence of SEQ ID NO: 95 (including a H at position36, S at a position 408 and Q at position 762; “36H 408S 762Qpolymerase”) was performed amplifying a 2 kb fragment of human genomicDNA.

dUTP replacement of dTTP (2 mM MgCl2). 2 kb fragment of human genomicDNA was amplified from 200 ng of human genomic DNA template in 50 µl PCRreactions in the presence of dATP, dCTP, and dGTP (each 200 µM) andvariable amounts of dUTP replacing dTTP (the final concentration of dUTPand dTTP was 200 µM) (FIG. 13 ). Primers with the following sequenceswere used:

GAAGAGCCAAGGACAGGTAC (SEQ ID NO: 64) (forward)

CCTCCAAATCAAGCCTCTAC (SEQ ID NO: 65) (reverse)

The PCR program was as follows:

98° C. 30 s 98° C. 10 s 61° C. 30 s x30 72° C. 1 min 72° C. 10 min

Products were detected by agarose gel electrophoresis and staining withethidium bromide. Detectable product was observed at up to when 60 µMdTTP was replaced with dUTP (FIG. 13 ).

dUTP replacement of dTTP (1.5 mM MgCl2). 5 kb fragment of human genomicDNA was amplified from 200 ng of human genomic DNA template in 50 µl PCRreactions in the presence of dATP, dCTP, and dGTP (each 200 µM) andvariable amounts of dUTP replacing dTTP (the final concentration of dUTPand dTTP was 200 µM) (FIG. 14 ). Primers with the following sequenceswere used:

CCAACATGGCGAAATGCTGT (SEQ ID NO: 170) (forward)

CATCAACAACACGGTCAGCC (SEQ ID NO: 171) (reverse)

The PCR program was as follows:

98° C. 30 s 98° C. 10 s 61° C. 30 s x30 72° C. 3 min 72° C. 10 min

Products were detected by agarose gel electrophoresis and staining withethidium bromide. Detectable product was observed at up to when 140 µMdTTP was replaced with dUTP (FIG. 14 ).

Additional dUTP. The PCR performance of the 36H 408S 762Q DNA polymeraseand thermophilic DNA polymerase with the sequence of SEQ ID NO: 169(including a H at position 36; “36H polymerase”) were compared byamplifying 2 kb PCR fragments from 200 ng of human genomic DNA templatein 50 µl PCR reactions in the presence of four standard dNTPs (each 200µM) and increasing amounts of dUTP (from 0 µM to 200 µM) (FIG. 15 ; the180 µM dUTP lane for the 36H 408S 762Q DNA polymerase appeared to havetechnical issues). The primers, PCR program, and product detection wereas described above for the 2kb human genomic fragment amplification inwhich dUTP replaced varying amounts of dTTP.

Easily detectable amounts of PCR product was observed even in thepresence of 200 µM dUTP for the 36H 408S 762Q DNA polymerase. It isbelieved that the 36H 408S 762Q DNA polymerase would still producedetectable amounts of PCR product in the presence of dUTP concentrationsof 220-260 µM. PCR products were not detected at 200 µM dUTP for 36H DNApolymerase.

The results showed that 36H 408S 762Q DNA polymerase can use dUTP as areplacement of dTTP in dNTPs mixture and has higher additional dUTPtolerance compared to 36H DNA polymerase.

Example 7. Performance of 36H 408S 762Q DNA Polymerase in MultiplexReactions and Tolerance of High Primer Concentration

The tolerance of high primer concentration of the 36H 408S 762Q DNApolymerase was assessed by amplifying regions of human gnomic DNAtemplate with 4 or 5 primer pairs in a multiplex PCR reaction. 8 ng ofhuman genomic DNA template was used in 20 µl PCR reactions. Total primerconcentration 5 µM to 100 µM was tested (FIG. 16 ). Primers with thefollowing sequences were used:

99 bp amplicon CCCACAGTTGGTAGGCATCA (SEQ ID NO: 172) (forward)TTGCTCAGCAACAAGTTGGC (SEQ ID NO: 173) (reverse) 131 bp ampliconTCATGTTGGACGGATGGCTG (SEQ ID NO: 174) (forward) CGGGCTGTCTTCATCACCTC(SEQ ID NO: 175) (reverse) 160 bp amplicon ACCATGTGAGACGCTAATCCA (SEQ IDNO: 176) (forward) ACCTGGGAGGCTTTTCTGTA (SEQ ID NO: 122) (reverse) 199bp amplicon GTTTATGGAGGTCCTCTTGTGTCC (SEQ ID NO: 123) (forward)GGGTCAACGCTAGGCTGGCAG (SEQ ID NO: 124) (reverse) 250 bp ampliconTCTGGACGGGCATCTCAAGT (SEQ ID NO: 125) (forward) TTCACAGGAAGCACTCACCA(SEQ ID NO: 126) (reverse)

The PCR program was as follows:

98° C. 30 s 98° C. 10 s 65° C. 10 s x30 72° C. 6 s

Products were analyzed by agarose gel electrophoresis and stained as inExample 6.

With both 4 and 5 primer pairs, all PCR products were detectable,indicating that the 36H 408S 762Q DNA polymerase tolerated high primerconcentrations (FIG. 16 ). Reasonably even amplification was observedusing multiple primer pairs, thus making this polymerase suitable forvarious multiplex PCR applications, for example, amplification of DNAfor next-generation sequencing.

Table of Sequences SEQ ID NO Description Sequence 1 Pfu DNA polymerase(GenBank Acc. No. WP_011011325.1) amino acid sequence MILDVDYITEEGKPVIRLFK KENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG KIVRIVDVEKVEKKFLGKPI TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY LIDKGLIPMEGEEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY VEVVSSEREMIKRFLRIIRE KDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAVEVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE SGENLERVAKYSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNEVAPNKPSEEEYQRR LRESYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVS PDTLNLEGCKNYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLLANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIPGGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLEIVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRPLHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDAEYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGLTSWL NIKKS 2 Pfu GenBankWP_011011325.1 R762X amino acid sequence MILDVDYITE EGKPVIRLFKKENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPITVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY LIDKGLIPME GEEELKILAFDIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDLYHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNEVAPN KPSEEEYQRRLRESYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYAKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLPAVLRILEGFG YRKEDLRYQK TXQVGLTSWL NIKKS; wherein X is selected from Q, N,H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 3 Pfu GenBank WP_011011325.1 R762Q amino acidsequence MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYA LLRDDSKIEEVKKITGERHG KIVRIVDVEK VEKKFLGKPI TVWKLYLEHP QDVPTIREKV REHPAVVDIFEYDIPFAKRY LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKVITWKNIDLPY VEVVSSEREM IKRFLRIIRE KDPDIIVTYN GDSFDFPYLA KRAEKLGIKLTIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKVYADEIAKAWE SGENLERVAK YSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTGNLVEWFLLRK AYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWEN IVYLDFRALYPSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKETQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELEEKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFVTKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQKLANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGPISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TQQVGLTSWL NIKKS4 Pfu GenBank WP_011011325.1 A408S R762X amino acid sequence MILDVDYITEEGKPVIRLFK KENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG KIVRIVDVEKVEKKFLGKPI TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY LIDKGLIPMEGEEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY VEVVSSEREMIKRFLRIIRE KDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAVEVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE SGENLERVAKYSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNEVAPNKPSEEEYQRR LRESYTGGFV KEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCKNYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLLANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIPGGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLEIVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRPLHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDAEYYIENQVLP AVLRILEGFG YRKEDLRYQK TXQVGLTSWL NIKKS; wherein X is selectedfrom Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in someembodiments, X is selected from Q and N. 5 Pfu GenBank WP_011011325.1A408S R762Q amino acid sequence MILDVDYITE EGKPVIRLFK KENGKFKIEHDRTFRPYIYA LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPI TVWKLYLEHPQDVPTIREKV REHPAVVDIF EYDIPFAKRY LIDKGLIPME GEEELKILAF DIETLYHEGEEFGKGPIIMI SYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIRE KDPDIIVTYNGDSFDFPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVITRTINLPTYTLEAVYE AIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATY ELGKEFLPMEIQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNEVAPN KPSEEEYQRR LRESYTGGFVKEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFIPSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKECAESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINSKLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLETILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAKGVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFGYRKEDLRYQK TQQVGLTSWL NIKKS 6 Pfu DNA polymerase (GenBank Acc. No.WP_011011325.1), catalytic domain amino acid sequence SYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEERQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TRQVGL7 Pfu DNA polymerase (GenBank Acc. No. WP_011011325.1) catalytic domainR762X amino acid sequence SYTGGFV KEPEKGLWEN IVYLDFRALY PSIIITHNVSPDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILLDYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYIDTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDEEGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQK TXQVGL; wherein X isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 8 Pfu DNA polymerase(GenBank Acc. No. WP_011011325.1) catalytic domain A408S R762X aminoacid sequence SYTGGFV KEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCKNYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLLANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIPGGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLEIVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRPLHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDAEYYIENQVLP AVLRILEGFG YRKEDLRYQK TXQVGL; wherein X is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, Xis selected from Q and N. 9 Pfu DNA polymerase (GenBank Acc. No.WP_011011325.1) catalytic domain R762Q amino acid sequence SYTGGFVKEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFIPSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKECAESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINSKLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLETILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAKGVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFGYRKEDLRYQK TQQVGL 10 Pfu DNA polymerase (GenBank Acc. No.WP_011011325.1) catalytic domain A408S R762Q amino acid sequence SYTGGFVKEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFIPSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKECAESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINSKLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLETILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAKGVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFGYRKEDLRYQK TQQVGL 11 Pfu GenBank WP_011011325.1 R762X with DNA bindingdomain amino acid sequence MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYALLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPI TVWKLYLEHP QDVPTIREKVREHPAVVDIF EYDIPFAKRY LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIRE KDPDIIVTYN GDSFDFPYLAKRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATY ELGKEFLPME IQLSRLVGQPLWDVSRSSTG NLVEWFLLRK AYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEERQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQKTXQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGKTGRGAVSEKD APKELLQMLE KQKK; wherein X is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, P, and G; in some embodiments, X is selectedfrom Q and N. 12 Pfu GenBank WP_011011325.1 R762Q with DNA bindingdomain amino acid sequence MILDVDYITE EGKPVIRLFK KENGKFKIEH DRTFRPYIYALLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPI TVWKLYLEHP QDVPTIREKVREHPAVVDIF EYDIPFAKRY LIDKGLIPME GEEELKILAF DIETLYHEGE EFGKGPIIMISYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIRE KDPDIIVTYN GDSFDFPYLAKRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVITRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATY ELGKEFLPME IQLSRLVGQPLWDVSRSSTG NLVEWFLLRK AYERNEVAPN KPSEEEYQRR LRESYTGGFV KEPEKGLWENIVYLDFRALY PSIIITHNVS PDTLNLEGCK NYDIAPQVGH KFCKDIPGFI PSLLGHLLEERQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGY AKARWYCKEC AESVTAWGRKYIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKK ALEFVKYINS KLPGLLELEYEGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIA KETQARVLET ILKHGDVEEAVRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKKLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRYQKTQQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGKTGRGAVSEKD APKELLQMLE KQKK 13 Pfu GenBank WP_011011325.1 A408S R762Xwith DNA binding domain amino acid sequence MILDVDYITE EGKPVIRLFKKENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG KIVRIVDVEK VEKKFLGKPITVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY LIDKGLIPME GEEELKILAFDIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY VEVVSSEREM IKRFLRIIREKDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDLYHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE SGENLERVAK YSMEDAKATYELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNEVAPN KPSEEEYQRRLRESYTGGFV KEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCK NYDIAPQVGHKFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLL ANSFYGYYGYAKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIP GGESEEIKKKALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLE IVRRDWSEIAKETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDA EYYIENQVLPAVLRILEGFG YRKEDLRYQK TXQVGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK; wherein X is selectedfrom Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in someembodiments, X is selected from Q and N. 14 Pfu GenBank WP_011011325.1A408S R762Q with DNA binding domain amino acid sequence MILDVDYITEEGKPVIRLFK KENGKFKIEH DRTFRPYIYA LLRDDSKIEE VKKITGERHG KIVRIVDVEKVEKKFLGKPI TVWKLYLEHP QDVPTIREKV REHPAVVDIF EYDIPFAKRY LIDKGLIPMEGEEELKILAF DIETLYHEGE EFGKGPIIMI SYADENEAKV ITWKNIDLPY VEVVSSEREMIKRFLRIIRE KDPDIIVTYN GDSFDFPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAVEVKGRIHFDL YHVITRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE SGENLERVAKYSMEDAKATY ELGKEFLPME IQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNEVAPNKPSEEEYQRR LRESYTGGFV KEPEKGLWEN IVYLDFRSLY PSIIITHNVS PDTLNLEGCKNYDIAPQVGH KFCKDIPGFI PSLLGHLLEE RQKIKTKMKE TQDPIEKILL DYRQKAIKLLANSFYGYYGY AKARWYCKEC AESVTAWGRK YIELVWKELE EKFGFKVLYI DTDGLYATIPGGESEEIKKK ALEFVKYINS KLPGLLELEY EGFYKRGFFV TKKRYAVIDE EGKVITRGLEIVRRDWSEIA KETQARVLET ILKHGDVEEA VRIVKEVIQK LANYEIPPEK LAIYEQITRPLHEYKAIGPH VAVAKKLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPKKHKYDAEYYIENQVLP AVLRILEGFG YRKEDLRYQK TQQVGLTSWL NIKKSGTGGG GATVKFKYKGEEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 15Pyrococcus K762X catalytic domain amino acid sequence SYAGGFVKEPEKGLWENIVS LDFRALYPSI IITHNVSPDT LNREGCRNYD VAPEVGHKFC KDFPGFIPSLLKRLLDERQK IKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKA RWYCKECAESVTAWGREYIE FVWKELEEKF GFKVLYIDTD GLYATIPGGK SEEIKKKALE FVDYINAKLPGLLELEYEGF YKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKET QARVLEAILKHGNVEEAVRI VKEVTQKLSK YEIPPEKLAI YEQITRPLHE YKAIGPHVAV AKRLAAKGVKIKPGMVIGYI VLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVL RILEGFGYRKEDLRWQKTXQ TGL; wherein X is selected from Q, N, H, S, T, Y, C, M, W, A,I, L, F, V, P, and G; in some embodiments, X is selected from Q and N.16 Pyrococcus A408S K762X catalytic domain amino acid sequenceSYAGGFVKEP EKGLWENIVS LDFRSLYPSI IITHNVSPDT LNREGCRNYD VAPEVGHKFCKDFPGFIPSL LKRLLDERQK IKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKARWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTD GLYATIPGGK SEEIKKKALEFVDYINAKLP GLLELEYEGF YKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKETQARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAI YEQITRPLHE YKAIGPHVAVAKRLAAKGVK IKPGMVIGYI VLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVLRILEGFGYRK EDLRWQKTXQ TGL; wherein X is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, P, and G; in some embodiments, X is selectedfrom Q and N. 17 Pyrococcus K762Q catalytic domain amino acid sequenceSYAGGFVKEP EKGLWENIVS LDFRALYPSI IITHNVSPDT LNREGCRNYD VAPEVGHKFCKDFPGFIPSL LKRLLDERQK IKTKMKASQD PIEKIMLDYR QRAIKILANS YYGYYGYAKARWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTD GLYATIPGGK SEEIKKKALEFVDYINAKLP GLLELEYEGF YKRGFFVTKK KYALIDEEGK IITRGLEIVR RDWSEIAKETQARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAI YEQITRPLHE YKAIGPHVAVAKRLAAKGVK IKPGMVIGYI VLRGDGPISN RAILAEEYDP RKHKYDAEYY IENQVLPAVLRILEGFGYRK EDLRWQKTQQ TGL 18 Pyrococcus 408S K762Q catalytic domainamino acid sequence SYAGGFVKEP EKGLWENIVS LDFRSLYPSI IITHNVSPDTLNREGCRNYD VAPEVGHKFC KDFPGFIPSL LKRLLDERQK IKTKMKASQD PIEKIMLDYRQRAIKILANS YYGYYGYAKA RWYCKECAES VTAWGREYIE FVWKELEEKF GFKVLYIDTDGLYATIPGGK SEEIKKKALE FVDYINAKLP GLLELEYEGF YKRGFFVTKK KYALIDEEGKIITRGLEIVR RDWSEIAKET QARVLEAILK HGNVEEAVRI VKEVTQKLSK YEIPPEKLAIYEQITRPLHE YKAIGPHVAV AKRLAAKGVK IKPGMVIGYI VLRGDGPISN RAILAEEYDPRKHKYDAEYY IENQVLPAVL RILEGFGYRK EDLRWQKTQQ TGL 76 Pyrococcus DNApolymerase sequence including exonuclease domain and catalytic domain,K762X MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYA LLKDDSKIEE VKKITAERHGKIVRIVDAEK VEKKFLGRPI TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMI SYADEEEAKV ITWKKIDLPYVEVVSSEREM IKRFLKIIRE KDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWETGEGLERVAK YSMEDAKATY ELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWEN IVSLDFRALY PSIIITHNVSPDTLNREGCR NYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVWKELE EKFGFKVLYIDTDGLYATIP GGKSEEIKKK ALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEA VRIVKEVTQK LSKYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TXQTGL; wherein X isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 77 Pyrococcus DNApolymerase sequence including exonuclease domain and catalytic domain,A408S K762X MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYA LLKDDSKIEEVKKITAERHG KIVRIVDAEK VEKKFLGRPI TVWRLYFEHP QDVPTIREKI REHSAVVDIFEYDIPFAKRY LIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMI SYADEEEAKVITWKKIDLPY VEVVSSEREM IKRFLKIIRE KDPDIIITYN GDSFDLPYLA KRAEKLGIKLTIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKVYADEIAKAWE TGEGLERVAK YSMEDAKATY ELGKEFFPME AQLSRLVGQP LWDVSRSSTGNLVEWFLLRK AYERNELAPN KPDEREYERR LRESYAGGFV KEPELGLWEN IVSLDFRSLYPSIIITHNVS PDTLNREGCR NYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQKIKTKMKASQDPIEKIML DYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVWKELEEKFGFKVLYI DTDGLYATIP GGKSEEIKKK ALEFVDYINA KLPGLLELEY EGFYKRGFFVTKKKYALIDE EGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEA VRIVKEVTQKLSKYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKRLAAK GVKIKPGMVI GYIVLRGDGPISNRAILAEE YDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TXQTGL; wherein Xis selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 78 Pyrococcus DNApolymerase sequence including exonuclease domain and catalytic domain,K762Q MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYA LLKDDSKIEE VKKITAERHGKIVRIVDAEK VEKKFLGRPI TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRYLIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMI SYADEEEAKV ITWKKIDLPYVEVVSSEREM IKRFLKIIRE KDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPKMQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWETGEGLERVAK YSMEDAKATY ELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRKAYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWEN IVSLDFRALY PSIIITHNVSPDTLNREGCR NYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIMLDYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVWKELE EKFGFKVLYIDTDGLYATIP GGKSEEIKKK ALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDEEGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEA VRIVKEVTQK LSKYEIPPEKLAIYEQITRP LHEYKAIGPH VAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEEYDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TQQTGL 79 Pyrococcus DNApolymerase sequence including exonuclease domain and catalytic domain,A408S K762Q MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYA LLKDDSKIEEVKKITAERHG KIVRIVDAEK VEKKFLGRPI TVWRLYFEHP QDVPTIREKI REHSAVVDIFEYDIPFAKRY LIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMI SYADEEEAKVITWKKIDLPY VEVVSSEREM IKRFLKIIRE KDPDIIITYN GDSFDLPYLA KRAEKLGIKLTIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKVYADEIAKAWE TGEGLERVAK YSMEDAKATY ELGKEFFPME AQLSRLVGQP LWDVSRSSTGNLVEWFLLRK AYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWEN IVSLDFRSLYPSIIITHNVS PDTLNREGCR NYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQKIKTKMKASQDPIEKIML DYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVWKELEEKFGFKVLYI DTDGLYATIP GGKSEEIKKK ALEFVDYINA KLPGLLELEY EGFYKRGFFVTKKKYALIDE EGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEA VRIVKEVTQKLSKYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKRLAAK GVKIKPGMVI GYIVLRGDGPISNRAILAEE YDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TQQTGL 19Pyrococcus DNA polymerase sequence including exonuclease domain and DNAbinding domain, K762X MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYALLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPI TVWRLYFEHP QDVPTIREKIREHSAVVDIF EYDIPFAKRY LIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMISYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIRE KDPDIIITYN GDSFDLPYLAKRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYEAIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATY ELGKEFFPME AQLSRLVGQPLWDVSRSSTG NLVEWFLLRK AYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWENIVSLDFRALY PSIIITHNVS PDTLNREGCR NYDVAPEVGH KFCKDFPGFI PSLLKRLLDERQKIKTKMKA SQDPIEKIML DYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGREYIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKK ALEFVDYINA KLPGLLELEYEGFYKRGFFV TKKKYALIDE EGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEAVRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKRLAAK GVKIKPGMVIGYIVLRGDGP ISNRAILAEE YDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQKTXQTGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGKTGRGAVSEKD APKELLQMLE KQKK; wherein X is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, P, and G; in some embodiments, X is selectedfrom Q and N. 20 Pyrococcus DNA polymerase sequence includingexonuclease domain and DNA binding domain, K762Q MILDADYITE EGKPVIRLFKKENGEFKIEH DRTFRPYIYA LLKDDSKIEE VKKITAERHG KIVRIVDAEK VEKKFLGRPITVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY LIDKGLIPME GDEELKLLAFDIETLYHEGE EFGKGPIIMI SYADEEEAKV ITWKKIDLPY VEVVSSEREM IKRFLKIIREKDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAV EVKGRIHFDLYHVIRRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE TGEGLERVAK YSMEDAKATYELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNELAPN KPDEREYERRLRESYAGGFV KEPEKGLWEN IVSLDFRALY PSIIITHNVS PDTLNREGCR NYDVAPEVGHKFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIML DYRQRAIKIL ANSYYGYYGYAKARWYCKEC AESVTAWGRE YIEFVWKELE EKFGFKVLYI DTDGLYATIP GGKSEEIKKKALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDE EGKIITRGLE IVRRDWSEIAKETQARVLEA ILKHGNVEEA VRIVKEVTQK LSKYEIPPEK LAIYEQITRP LHEYKAIGPHVAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPRKHKYDA EYYIENQVLPAVLRILEGFG YRKEDLRWQK TQQTGLTSWL NIKKSGTGGG GATVKFKYKG EEKEVDISKIKKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 21 Pyrococcus DNApolymerase sequence including exonuclease domain and DNA binding domain,A408S K762X MILDADYITE EGKPVIRLFK KENGEFKIEH DRTFRPYIYA LLKDDSKIEEVKKITAERHG KIVRIVDAEK VEKKFLGRPI TVWRLYFEHP QDVPTIREKI REHSAVVDIFEYDIPFAKRY LIDKGLIPME GDEELKLLAF DIETLYHEGE EFGKGPIIMI SYADEEEAKVITWKKIDLPY VEVVSSEREM IKRFLKIIRE KDPDIIITYN GDSFDLPYLA KRAEKLGIKLTIGRDGSEPK MQRIGDMTAV EVKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKVYADEIAKAWE TGEGLERVAK YSMEDAKATY ELGKEFFPME AQLSRLVGQP LWDVSRSSTGNLVEWFLLRK AYERNELAPN KPDEREYERR LRESYAGGFV KEPEKGLWEN IVSLDFRSLYPSIIITHNVS PDTLNREGCR NYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQKIKTKMKASQDPIEKIML DYRQRAIKIL ANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVWKELEEKFGFKVLYI DTDGLYATIP GGKSEEIKKK ALEFVDYINA KLPGLLELEY EGFYKRGFFVTKKKYALIDE EGKIITRGLE IVRRDWSEIA KETQARVLEA ILKHGNVEEA VRIVKEVTQKLSKYEIPPEK LAIYEQITRP LHEYKAIGPH VAVAKRLAAK GVKIKPGMVI GYIVLRGDGPISNRAILAEE YDPRKHKYDA EYYIENQVLP AVLRILEGFG YRKEDLRWQK TXQTGLTSWLNIKKSGTGGG GATVKFKYKG EEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKDAPKELLQMLE KQKK; wherein X is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, P, and G; in some embodiments, X is selected from Q andN. 22 Pyrococcus DNA polymerase sequence including exonuclease domainand sequence non-specific DNA binding domain, A408S K762Q MILDADYITEEGKPVIRLFK KENGEFKIEH DRTFRPYIYA LLKDDSKIEE VKKITAERHG KIVRIVDAEKVEKKFLGRPI TVWRLYFEHP QDVPTIREKI REHSAVVDIF EYDIPFAKRY LIDKGLIPMEGDEELKLLAF DIETLYHEGE EFGKGPIIMI SYADEEEAKV ITWKKIDLPY VEVVSSEREMIKRFLKIIRE KDPDIIITYN GDSFDLPYLA KRAEKLGIKL TIGRDGSEPK MQRIGDMTAVEVKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKV YADEIAKAWE TGEGLERVAKYSMEDAKATY ELGKEFFPME AQLSRLVGQP LWDVSRSSTG NLVEWFLLRK AYERNELAPNKPDEREYERR LRESYAGGFV KEPEKGLWEN IVSLDFRSLY PSIIITHNVS PDTLNREGCRNYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQKIKTKMKA SQDPIEKIML DYRQRAIKILANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVWKELE EKFGFKVLYI DTDGLYATIPGGKSEEIKKK ALEFVDYINA KLPGLLELEY EGFYKRGFFV TKKKYALIDE EGKIITRGLEIVRRDWSEIA KETQARVLEA ILKHGNVEEA VRIVKEVTQK LSKYEIPPEK LAIYEQITRPLHEYKAIGPH VAVAKRLAAK GVKIKPGMVI GYIVLRGDGP ISNRAILAEE YDPRKHKYDAEYYIENQVLP AVLRILEGFG YRKEDLRWQK TQQTGLTSWL NIKKSGTGGG GATVKFKYKGEEKEVDISKI KKVWRVGKMI SFTYDEGGGK TGRGAVSEKD APKELLQMLE KQKK 23 K762Xvariant of Deep Vent DNA polymerase amino acid sequence MILDADYITEDGKPIIRIFK KENGEFKVEY DRNFRPYIYA LLKDDSQIDE VRKITAERHG KIVRIIDAEKVRKKFLGRPI EVWRLYFEHP QDVPAIRDKI REHSAVIDIF EYDIPFAKRY LIDKGLIPMEGDEELKLLAF DIETLYHEGE EFAKGPIIMI SYADEEEAKV ITWKKIDLPY VEVVSSEREMIKRFLKVIRE KDPDVIITYN GDSFDLPYLV KRAEKLGIKL PLGRDGSEPK MQRLGDMTAVEIKGRIHFDL YHVIRRTINL PTYTLEAVYE AIFGKPKEKV YAHEIAEAWE TGKGLERVAKYSMEDAKVTY ELGREFFPME AQLSRLVGQP LWDVSRSSTG NLVEWYLLRK AYERNELAPNKPDEREYERR LRESYAGGYV KEPEKGLWEG LVSLDFRSLY PSIIITHNVS PDTLNREGCREYDVAPEVGH KFCKDFPGFI PSLLKRLLDE RQEIKRKMKA SKDPIEKKML DYRQRAIKILANSYYGYYGY AKARWYCKEC AESVTAWGRE YIEFVRKELE EKFGFKVLYI DTDGLYATIPGAKPEEIKKK ALEFVDYINA KLPGLLELEY EGFYVRGFFV TKKKYALIDE EGKIITRGLEIVRRDWSEIA KETQAKVLEA ILKHGNVEEA VKIVKEVTEK LSKYEIPPEK LVIYEQITRPLHEYKAIGPH VAVAKRLAAR GVKVRPGMVI GYIVLRGDGP ISKRAILAEE FDLRKHKYDAEYYIENQVLP AVLRILEAFG YRKEDLRWQK TXQTGLTAWL NIKKK; wherein X is selectedfrom Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in someembodiments, X is selected from Q and N. 24 K762Q variant of Deep VentDNA polymerase amino acid sequence MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPMEAYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELE ALEFVDYINAEGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TQQTGLTAWL DGKPIIRIFKVRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDLYAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKKKENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG DRNFRPYIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIREPLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGHSKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISKRAILAEE YRKEDLRWQK 25 K762X variant of Deep Vent DNA polymerasecatalytic domain amino acid sequence SYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK KFCKDFPGFI DYRQRAIKILYIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDATXQTGL; PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKVRPGMVI EYYIENQVLP wherein X is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 26 K762Q variant of Deep Vent DNA polymerasecatalytic domain amino acid sequence SYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK KFCKDFPGFI DYRQRAIKILYIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TQQTGLPSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEKGVKVRPGMVI EYYIENQVLP 27 K762X variant of Deep Vent DNA polymerase aminoacid sequence with DNA binding domain MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPMEAYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL DGKPIIRIFK VRKITAERHGQDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY KENGEFKVEYKIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINLTGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKECDRNFRPYIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKMLAESVTAWGRE YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAARFDLRKHKYDA TXQTGLTAWL KKVWRVGKMI KQKK; EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKKGTGGG SFTYDEGGGK DTDGLYATIPEGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKGTGRGAVSEKD GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEEYRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 28 K762Q variant of Deep Vent DNA polymeraseamino acid sequence with DNA binding domain MILDADYITE LLKDDSQIDEEVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKVELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELEALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TQQTGLTAWLKKVWRVGKMI KQKK DGKPIIRIFK VRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPYGDSFDLPYLV EIKGRIHFDL YAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVSPSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEKGVKVRPGMVI EYYIENQVLP NIKKKGTGGG SFTYDEGGGK KENGEFKVEY KIVRIIDAEKREHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAKLWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIPEGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKGTGRGAVSEKD DRNFRPYIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIREPLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGHSKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISKRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLE 29 K762X K775S variant ofDeep Vent DNA polymerase amino acid sequence with sequence non-specificDNA binding domain MILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPMESYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPME AYERNELAPNLVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELE ALEFVDYINA EGKIITRGLEVKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TXQTGLTAWL KKVWRVGKMI KQKK; DGKPIIRIFKVRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDLYAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLPNIKKSGTGGG SFTYDEGGGK KENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYVPDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEALVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKG TGRGAVSEKD DRNFRPYIYAVRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYEYSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGREGAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQKEEKEVDISKI APKELLQMLE wherein X is selected from Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, P, and G; in some embodiments, X is selected from Qand N. 30 K762Q K775S variant of Deep Vent DNA polymerase amino acidsequence with sequence MILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPMEDGKPIIRIFK VRKITAERHG QDVPAIRDKI GDEELKLLAF KENGEFKVEY KIVRIIDAEKREHSAVIDIF DIETLYHEGE DRNFRPYIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMInon-specific DNA binding domain SYADEEEAKV KDPDVIITYN MQRLGDMTAVAIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKILYIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDATQQTGLTAWL KKVWRVGKMI KQKK ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKSGTGGGSFTYDEGGGK VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAK LWDVSRSSTGLRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIP EGFYVRGFFVKETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKG TGRGAVSEKDIKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEGEYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEALHEYKAIGPH ISKRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLE 31 K764X variantof Thermococcus litoralis DNA polymerase MILDTDYITK LLKDDSAIEEEVWKLIFEHP LIDKGLIPME SYADEEEARV KDPDVIITYN PKIQRMGDSF YEAVLGKTKSTYELGKEFFP RVAYARNELA ENIIYLDFRS GYRFCKDFPG MLDYRQRAIK RHYIEMTIREKKAKEFLNYI DEEGRITTRG KAVEVVRDVV PHVAIAKRLA TEYDPRKHKY QSSXQTGLDADGKPIIRIFK IKAIKGERHG QDVPAMRGKI GDEELKLLAF ITWKNIDLPY GDNFDLPYLIAVEIKGRIHF KLGAEEIAAI MEAELAKLIG PNKPDEEEYK LYPSIIVTHN FIPSILGDLILLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGTDPDYYIENQV WLKR; KENGEFKIEL KTVRVLDAVK REHPAVVDIY DIETFYHEGD VDVVSNEREMKRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSS RRLRTTYLGG VSPDTLEKEGAMRQDIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQITIISYIVLKGS LPAVLRILEA DPHFQPYIYA VRKKFLGREV EYDIPFAKRY EFGKGEIIMIIKRFVQVVKE VLGRDKEHPE NLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLWCKNYDVAPIV KSTIDPIEKK ECAESVTAWG IPGEKPELIK FVTKKRYAVI EAILKEGSVERDLKDYKAIG GKISDRVILL FGYRKEDLRY wherein X is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 32 K764Q variant of Thermococcus litoralis DNApolymerase MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARVKDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYARNELA ENIIYLDFRSGYRFCKDFPG MLDYRQRAIK RHYIEMTIRE KKAKEFLNYI DEEGRITTRG KAVEVVRDVVPHVAIAKRLA TEYDPRKHKY QSSQQTGLDA DGKPIIRIFK IKAIKGERHG QDVPAMRGKIGDEELKLLAF ITWKNIDLPY GDNFDLPYLI AVEIKGRIHF KLGAEEIAAI MEAELAKLIGPNKPDEEEYK LYPSIIVTHN FIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLELLEVVRRDWSE EKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR KENGEFKIEL KTVRVLDAVKREHPAVVDIY DIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTI WETEESMKKLQSVWDVSRSS RRLRTTYLGG VSPDTLEKEG AMRQDIKKKM GYPKARWYSK YADTDGFYATEYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA DPHFQPYIYAVRKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKE VLGRDKEHPE NLPTYTLEAVAQYSMEDARA TGNLVEWYLL YVKEPEKGLW CKNYDVAPIV KSTIDPIEKK ECAESVTAWGIPGEKPELIK FVTKKRYAVI EAILKEGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRY 33K764X variant of Thermococcus litoralis DNA TYLGG VSPDTLEKEG CKNYDVAPIVGYRFCKDFPG FIPSILGDLI polymerase catalytic domain amino acid sequenceAMRQDIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQITIISYIVLKGS LPAVLRILEA KSTIDPIEKK ECAESVTAWG IPGEKPELIK FVTKKRYAVIEAILKEGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRY MLDYRQRAIK RHYIEMTIREKKAKEFLNYI DEEGRITTRG KAVEVVRDVV PHVAIAKRLA TEYDPRKHKY QSSXQTGL;LLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGTDPDYYIENQV wherein X is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X is selected from Q and N. 34K764Q variant of Thermococcus litoralis DNA polymerase catalytic domainamino acid sequence TYLGG VSPDTLEKEG AMRQDIKKKM GYPKARWYSK YADTDGFYATEYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA CKNYDVAPIVKSTIDPIEKK ECAESVTAWG IPGEKPELIK FVTKKRYAVI EAILKEGSVE RDLKDYKAIGGKISDRVILL FGYRKEDLRY GYRFCKDFPG MLDYRQRAIK RHYIEMTIRE KKAKEFLNYIDEEGRITTRG KAVEVVRDVV PHVAIAKRLA TEYDPRKHKY QSSQQTGL FIPSILGDLILLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGTDPDYYIENQV 35 K764X variant of Thermococcus litoralis DNA polymerase,sequence 2 (acc. ADK47977.1) MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPMESYADEEEARV KDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYERNELAENIIYLDFRS SYRFCKDFPG MLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRGKAVEIVRDVL PHVAIAKRLA TEYDPEKHKY QSSXQTGLDA DGKPIIRIFK IKAIKGERHGQDVPAMRDKI GDEELKLLAF ITWKNIDLPY GDNFDLPYLI AVEIKGRIHF KLGAEEIAAIMEAELAKLIG PNKPDEEEYK LYPSIIVTHN FIPSILGDLI LLANSYYGYM IEEKFGFKVLNSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR; KENGEFKIELKSVRVVDAVK KEHPAVIDIY DIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTIWETEESMKKL QSVWDVSRSS RRLRTTYLGG VSPDTLEKEG AMRQEIKKKM GYPKARWYSKYADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEADPHFQPYIYA VKKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKE VLGRDKENPENLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLW CENYDIAPIV KATIDPVERKECAESVTAWG ISGEKPEIIK FVTKKRYAVI EAILKDGSVE RDLKDYKAIG GKISDRVILLFGYRKEDLRY wherein X is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X is selected from Q and N. 36K764Q variant of Thermococcus litoralis DNA polymerase, sequence 2 (acc.ADK47977.1) MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARVKDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYERNELA ENIIYLDFRSSYRFCKDFPG MLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRG KAVEIVRDVLPHVAIAKRLA DGKPIIRIFK IKAIKGERHG QDVPAMRDKI GDEELKLLAF ITWKNIDLPYGDNFDLPYLI AVEIKGRIHF KLGAEEIAAI MEAELAKLIG PNKPDEEEYK LYPSIIVTHNFIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPLARGIKVKPGT KENGEFKIEL KSVRVVDAVK KEHPAVIDIY DIETFYHEGD VDVVSNEREMKRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSS RRLRTTYLGG VSPDTLEKEGAMRQEIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQITIISYIVLKGS DPHFQPYIYA VKKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKEVLGRDKENPE NLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLW CENYDIAPIVKATIDPVERK ECAESVTAWG ISGEKPEIIK FVTKKRYAVI EAILKDGSVE RDLKDYKAIGGKISDRVILL TEYDPEKHKY QSSQQTGLDA DPDYYIENQV WLKR LPAVLRILEA FGYRKEDLRY37 K764X variant of Thermococcus litoralis DNA polymerase, sequence 2(acc. ADK47977.1), catalytic domain amino acid sequence TYLGG VSPDTLEKEGAMRQEIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQITIISYIVLKGS LPAVLRILEA CENYDIAPIV KATIDPVERK ECAESVTAWG ISGEKPEIIKFVTKKRYAVI EAILKDGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRY SYRFCKDFPGMLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRG KAVEIVRDVL PHVAIAKRLATEYDPEKHKY QSSXQTGL; FIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLELLEVVRRDWSE EKIAKYRVPL ARGIKVKPGT DPDYYIENQV wherein X is selected fromQ, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments,X is selected from Q and N. 38 K764Q variant of Thermococcus litoralisDNA polymerase, sequence 2 (acc. ADK47977.1), catalytic domain aminoacid sequence TYLGG VSPDTLEKEG AMRQEIKKKM GYPKARWYSK YADTDGFYATEYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA CENYDIAPIVKATIDPVERK ECAESVTAWG ISGEKPEIIK FVTKKRYAVI EAILKDGSVE RDLKDYKAIGGKISDRVILL FGYRKEDLRY SYRFCKDFPG MLDYRQRAVK RHYIEMTIKE KKAREFLNYIDEEGRITTRG KAVEIVRDVL PHVAIAKRLA TEYDPEKHKY QSSQQTGL FIPSILGDLILLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGTDPDYYIENQV 39 R761X variant of Thermococcus gorgonarius DNA polymeraseMILDTDYITE LLKDDSAIED EVWKLYFTHP LIDKGLIPME SYADEEGARV KDPDVLITYNIQRMGDRFAV AIFGQPKEKV ELGKEFFPME AYERNELAPN VYLDFRSLYP FCKDFPGFIPYRQRAIKILA IETTIREIEE KEFLDYINAK DKITTRGLEI RIVKEVTEKL AVAKRLAARGDPAKHKYDAE XQVGLGAWLK DGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAFITWKNIDLPY GDNFDFAYLK EVKGRIHFDL YAEEIAQAWE AQLSRLVGQS KPDERELARRSIIITHNVSP SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAKSKYEVPPEKL IKIRPGTVIS YYIENQVLPA PKT; KENGEFKIDY TTVRVVRAEK KEHPAVVDIYDIETLYHEGE VDVVSTEKEM KRSEKLGVKF YPVIRRTINL TGEGLERVAR LWDVSRSSTGRESYAGGYVK DTLNREGCEE QKVKKKMKAT KARWYCKECA TDGFFATIPG GFYKRGFFVTETQARVLEAI VIYEQITRDL YIVLKGSGRI VERILRAFGY DRNFEPYIYA VKKKFLGRPIEYDIPFAKRY EFAEGPILMI IKRFLKVVKE ILGREGSEPK PTYTLEAVYE YSMEDAKVTYNLVEWFLLRK EPERGLWENI YDVAPQVGHK IDPIEKKLLD ESVTAWGRQY ADAETVKKKAKKKYAVIDEE LKHGDVEEAV KDYKATGPHV GDRAIPFDEF RKEDLRYQKT wherein X isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X is selected from Q and N. 40 R761Q variant ofThermococcus gorgonarius DNA polymerase MILDTDYITE LLKDDSAIED EVWKLYFTHPLIDKGLIPME SYADEEGARV KDPDVLITYN IQRMGDRFAV AIFGQPKEKV ELGKEFFPMEAYERNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILA IETTIREIEE KEFLDYINAKDGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAF ITWKNIDLPY GDNFDFAYLKEVKGRIHFDL YAEEIAQAWE AQLSRLVGQS KPDERELARR SIIITHNVSP SLLGDLLEERNSFYGYYGYA KFGFKVLYAD LPGLLELEYE KENGEFKIDY TTVRVVRAEK KEHPAVVDIYDIETLYHEGE VDVVSTEKEM KRSEKLGVKF YPVIRRTINL TGEGLERVAR LWDVSRSSTGRESYAGGYVK DTLNREGCEE QKVKKKMKAT KARWYCKECA TDGFFATIPG GFYKRGFFVTDRNFEPYIYA VKKKFLGRPI EYDIPFAKRY EFAEGPILMI IKRFLKVVKE ILGREGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENI YDVAPQVGHK IDPIEKKLLDESVTAWGRQY ADAETVKKKA KKKYAVIDEE DKITTRGLEI RIVKEVTEKL AVAKRLAARGDPAKHKYDAE QQVGLGAWLK VRRDWSEIAK SKYEVPPEKL IKIRPGTVIS YYIENQVLPA PKTETQARVLEAI VIYEQITRDL YIVLKGSGRI VERILRAFGY LKHGDVEEAV KDYKATGPHVGDRAIPFDEF RKEDLRYQKT 41 R761X variant of Thermococcus gorgonarius DNApolymerase, catalytic domain amino acid sequence SYAGGYVK DTLNREGCEEQKVKKKMKAT KARWYCKECA TDGFFATIPG GFYKRGFFVT ETQARVLEAI VIYEQITRDLYIVLKGSGRI VERILRAFGY YDVAPQVGHK IDPIEKKLLD ESVTAWGRQY ADAETVKKKAKKKYAVIDEE LKHGDVEEAV KDYKATGPHV GDRAIPFDEF RKEDLRYQKT FCKDFPGFIPYRQRAIKILA IETTIREIEE KEFLDYINAK DKITTRGLEI RIVKEVTEKL AVAKRLAARGDPAKHKYDAE XQVGL; SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAKSKYEVPPEKL IKIRPGTVIS YYIENQVLPA wherein X is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 42 R761Q variant of Thermococcus gorgonarius DNApolymerase, catalytic domain amino acid sequence SYAGGYVK VYLDFRSLYPDTLNREGCEE QKVKKKMKAT KARWYCKECA TDGFFATIPG GFYKRGFFVT ETQARVLEAIVIYEQITRDL YIVLKGSGRI VERILRAFGY YDVAPQVGHK IDPIEKKLLD ESVTAWGRQYADAETVKKKA KKKYAVIDEE LKHGDVEEAV KDYKATGPHV GDRAIPFDEF RKEDLRYQKTFCKDFPGFIP YRQRAIKILA IETTIREIEE KEFLDYINAK DKITTRGLEI RIVKEVTEKLAVAKRLAARG DPAKHKYDAE QQVGL SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYEVRRDWSEIAK SKYEVPPEKL IKIRPGTVIS YYIENQVLPA 43 R761X variant ofThermococcus kodakarensis DNA polymerase MILDTDYITE LLKDDSAIEEEVWKLYFTHP LIDKGLVPME SYADEEGARV KDPDVLITYN IQRMGDRFAV AVFGQPKEKVELGKEFLPME AYERNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILA ITMTIKEIEEMEFLKYINAK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHKYDAE XQVGLSAWLKDGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAF ITWKNVDLPY GDNFDFAYLKEVKGRIHFDL YAEEITTAWE AQLSRLIGQS KPDEKELARR SIIITHNVSP SLLGDLLEERNSYYGYYGYA KYGFKVIYSD LPGALELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVISYYIENQVLPA PKGT; KENGEFKIEY TVVTVKRVEK REHPAVIDIY DIETLYHEGE VDVVSTEREMKRCEKLGINF YPVIRRTINL TGENLERVAR LWDVSRSSTG RQSYEGGYVK DTLNREGCKEQKIKKKMKAT RARWYCKECA TDGFFATIPG GFYKRGFFVT ETQARVLEAL VIHEQITRDLYIVLKGSGRI VERILRAFGY DRTFEPYFYA VQKKFLGRPV EYDIPFAKRY EFAEGPILMIIKRFLRVVKE ALGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENIYDVAPQVGHR IDPIERKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKDGDVEKAVKDYKATGPHV GDRAIPFDEF RKEDLRYQKT wherein X is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 44 R761Q variant of Thermococcus kodakarensis DNApolymerase MILDTDYITE LLKDDSAIEE EVWKLYFTHP LIDKGLVPME SYADEEGARVKDPDVLITYN IQRMGDRFAV AVFGQPKEKV ELGKEFLPME AYERNELAPN VYLDFRSLYPFCKDFPGFIP DGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAF ITWKNVDLPYGDNFDFAYLK EVKGRIHFDL YAEEITTAWE AQLSRLIGQS KPDEKELARR SIIITHNVSPSLLGDLLEER KENGEFKIEY TVVTVKRVEK REHPAVIDIY DIETLYHEGE VDVVSTEREMKRCEKLGINF YPVIRRTINL TGENLERVAR LWDVSRSSTG RQSYEGGYVK DTLNREGCKEQKIKKKMKAT DRTFEPYFYA VQKKFLGRPV EYDIPFAKRY EFAEGPILMI IKRFLRVVKEALGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENI YDVAPQVGHRIDPIERKLLD YRQRAIKILA ITMTIKEIEE MEFLKYINAK GKITTRGLEI RIVKEVTEKLAVAKRLAARG DPTKHKYDAE QQVGLSAWLK NSYYGYYGYA KYGFKVIYSD LPGALELEYEVRRDWSEIAK SKYEVPPEKL VKIRPGTVIS YYIENQVLPA PKGT RARWYCKECA TDGFFATIPGGFYKRGFFVT ETQARVLEAL VIHEQITRDL YIVLKGSGRI VERILRAFGY ESVTAWGREYADAETVKKKA KKKYAVIDEE LKDGDVEKAV KDYKATGPHV GDRAIPFDEF RKEDLRYQKT 45R761X variant of Thermococcus kodakarensis DNA polymerase, catalyticdomain amino acid sequence SYEGGYVK DTLNREGCKE QKIKKKMKAT RARWYCKECATDGFFATIPG GFYKRGFFVT ETQARVLEAL VIHEQITRDL YIVLKGSGRI VERILRAFGYYDVAPQVGHR IDPIERKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKDGDVEKAVKDYKATGPHV GDRAIPFDEF RKEDLRYQKT FCKDFPGFIP YRQRAIKILA ITMTIKEIEEMEFLKYINAK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHKYDAE XQVGL; SLLGDLLEERNSYYGYYGYA KYGFKVIYSD LPGALELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVISYYIENQVLPA wherein X is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X is selected from Q and N. 46R761Q variant of Thermococcus kodakarensis DNA polymerase, catalyticdomain amino acid sequence SYEGGYVK DTLNREGCKE QKIKKKMKAT RARWYCKECATDGFFATIPG GFYKRGFFVT ETQARVLEAL VIHEQITRDL YIVLKGSGRI VERILRAFGYYDVAPQVGHR IDPIERKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKDGDVEKAVKDYKATGPHV GDRAIPFDEF RKEDLRYQKT FCKDFPGFIP YRQRAIKILA ITMTIKEIEEMEFLKYINAK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHKYDAE QQVGL SLLGDLLEERNSYYGYYGYA KYGFKVIYSD LPGALELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVISYYIENQVLPA 47 K761X variant of Thermococcus species 9°N-7 DNApolymerase, catalytic domain amino acid sequence GYAGGYVK DTLNREGCKEQKIKRKMKAT KARWYCKECA TDGLHATIPG GFYVRGFFVT ETQARVLEAI VIHEQITRDLYIVLKGSGRI VERILKAFGY YDVAPEVGHK VDPLEKKLLD ESVTAWGREY ADAETVKKKAKKKYAVIDEE LKHGDVEEAV RDYKATGPHV GDRAIPADEF RKEDLRYQKT FCKDFPGFIPYRQRAIKILA IEMVIRELEE KEFLKYINPK GKITTRGLEI RIVKEVTEKL AVAKRLAARGDPTKHRYDAE XQVGL; SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAKSKYEVPPEKL VKIRPGTVIS YYIENQVLPA wherein X is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X isselected from Q and N. 48 K761Q variant of Thermococcus species 9°N-7DNA polymerase, catalytic domain amino acid sequence GYAGGYVK DTLNREGCKEQKIKRKMKAT KARWYCKECA TDGLHATIPG GFYVRGFFVT ETQARVLEAI VIHEQITRDLYIVLKGSGRI VERILKAFGY YDVAPEVGHK VDPLEKKLLD ESVTAWGREY ADAETVKKKAKKKYAVIDEE LKHGDVEEAV RDYKATGPHV GDRAIPADEF RKEDLRYQKT FCKDFPGFIPYRQRAIKILA IEMVIRELEE KEFLKYINPK GKITTRGLEI RIVKEVTEKL AVAKRLAARGDPTKHRYDAE QQVGL SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAKSKYEVPPEKL VKIRPGTVIS YYIENQVLPA 49 K761X variant of Thermococcusspecies 9°N-7 DNA polymerase MILDTDYITE LLKDDSAIED EVWKLYFNHP LIDKGLIPMESYADGSEARV KDPDVLITYN IQRMGDRFAV AVFGKPKEKV ELGREFFPME NGKPVIRVFKVKKVTAKRHG QDVPAIRDRI GDEELTMLAF ITWKKIDLPY GDNFDFAYLK EVKGRIHFDLYAEEIAQAWE AQLSRLIGQS KENGEFKIEY TVVKVKRAEK RAHPAVVDIY DIETLYHEGEVDVVSTEKEM KRCEELGIKF YPVIRRTINL SGEGLERVAR LWDVSRSSTG DRTFEPYFYAVQKKFLGRPI EYDIPFAKRY EFGTGPILMI IKRFLRVVRE TLGRDGSEPK PTYTLEAVYEYSMEDAKVTY NLVEWFLLRK AYKRNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILAIEMVIRELEE KEFLKYINPK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHRYDAEXQVGLGAWLK KPDERELARR SIIITHNVSP SLLGDLLEER NSFYGYYGYA KFGFKVLYADLPGLLELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVIS YYIENQVLPA VKGKK; RGGYAGGYVKDTLNREGCKE QKIKRKMKAT KARWYCKECA TDGLHATIPG GFYVRGFFVT ETQARVLEAIVIHEQITRDL YIVLKGSGRI VERILKAFGY EPERGLWDNI YDVAPEVGHK VDPLEKKLLDESVTAWGREY ADAETVKKKA KKKYAVIDEE LKHGDVEEAV RDYKATGPHV GDRAIPADEFRKEDLRYQKT wherein X is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X is selected from Q and N. 50K761Q variant of Thermococcus species 9°N-7 DNA polymerase MILDTDYITELLKDDSAIED EVWKLYFNHP LIDKGLIPME SYADGSEARV KDPDVLITYN IQRMGDRFAVAVFGKPKEKV ELGREFFPME AYKRNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILAIEMVIRELEE KEFLKYINPK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHRYDAEQQVGLGAWLK NGKPVIRVFK VKKVTAKRHG QDVPAIRDRI GDEELTMLAF ITWKKIDLPYGDNFDFAYLK EVKGRIHFDL YAEEIAQAWE AQLSRLIGQS KPDERELARR SIIITHNVSPSLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAK SKYEVPPEKLVKIRPGTVIS YYIENQVLPA VKGKK KENGEFKIEY TVVKVKRAEK RAHPAVVDIY DIETLYHEGEVDVVSTEKEM KRCEELGIKF YPVIRRTINL SGEGLERVAR LWDVSRSSTG RGGYAGGYVKDTLNREGCKE QKIKRKMKAT KARWYCKECA TDGLHATIPG GFYVRGFFVT ETQARVLEAIVIHEQITRDL YIVLKGSGRI VERILKAFGY DRTFEPYFYA VQKKFLGRPI EYDIPFAKRYEFGTGPILMI IKRFLRVVRE TLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRKEPERGLWDNI YDVAPEVGHK VDPLEKKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEELKHGDVEEAV RDYKATGPHV GDRAIPADEF RKEDLRYQKT 51 E775Q variant ofPyrobaculum calidifontis DNA polymerase MRFWPLDATY YFYAKCDKCD FLKVVAKVPEIDKGVVPCAW PPLRVLAFDI FEAEGRDDRR SERAKALGVP IVDEFPEIKV NDPAKRPTLMPLDQVAAASV KGAIVLEPKP EPHEPDPPEG RAVREEAKKY VGARWYKKEV TDSLFVKKSGAKKRYAGLLR ILKSKSVGEA LDKELDEYKA GPGKVSERAM VLGVKESDLK SVVGGVPEVRASLAKSYLSR DVRKLREAAL NVVEAREAGK EVYNERGSPD VIRGFVDFVK LRVDRLGGVPKTLDRVAEYF RYVLDDVRST GNRVEWMLLR GLYSDVLVLD VVVAPEVGHR PPDSPEYRLLAESVTAFARA AVDRLVKYVE DGRIDIVGFE RERVVKYVRE YGPHVHAALE PYIFVDDASKTGRVQKSLLD VFGVDGEGRR VAPVEAVEVV GAPGVVDVYE LGPLPLYEVV PLRDPVVMLAEFDPDVIVGY QQSVYGHWSV GVMKRSERVL LGLAEKLLPF YAYRMGEVAP FSSMYPNIMMFRKAPTGFIP DERQRALKVM ILLDVVEYAK ERHGIEIKVD VVRGDWCELA VVERLKAYKFLKRRGYKVGK VDVDYYIEKQ FLG VVLVDRRFRP ERRFFGRPTI ADIRYYMRYM EWAGVEEGFPVKTSDGREEV NSNGFDWPYL VGRANVDLYN IPGHKVYEYW LIQLSSVSGL NREEREYEPYKYNLSPDTYL AVLKHLVELR ANAMYGYLGW RLGIEVIYGD KDYERVLFTE KEVQLNVVELDLDDLIIWKT GTTVGYVIVR VIPAALRIAE 52 E778Q variant of Pyrobaculumaerophilum DNA polymerase MKFKLWPLDA RPYFYADCPA RSFLKIVARV YMLDMGVVPCGFPPPLRVLA VEVFEASGRD PYLAERARAL LYNIVDEFPE EYWRDQGKRP TYSVVGGVPECDPESVRSQL PEDVRKLREA SWNTVDAEAT FDIEVYNERG DRSVLRSFID GIPLKVDRVGIKLKTLDRVA LLRQYVIDDV VRIFGISESG GRVAPVEEVV AAALPGVSGV GEKLGNLPVYTPDPLRDPVI FVREFDPDVI GAPQQSVYGH EYFGVMKREE KSTYGLAEKL DRVVVVDRRFAVERRYLGRP YEADIRFYMR KVAEWGGVTE LLAVQASDGR VGYNSNQFDW WSVTGRANVDRVLVPGHKIY LPFLIQLSSV SGLPLDQVAA EPYKGAIVLE TYLERGEPDP ELRKRVREELTGWVGARWYK YGDTDSLFVK FTEAKKRYAG IELILTSRDV WKTLDKELDE VVKGGEKVSEIAEVIGIKEG ASVGNRVEWM PRPGLYSDVL PGGVYVAPEV KKYPPDSPEY KEVAESVTAFKSGDVEKLVK LLRDGRIDIV SEARQKVVKY YKAYPPHVHA RAVPYIFIDD DLKTGRSQRTLLRYAYRLGE ALDFSSMYPN GHRFRREPPG RVLDERQRAL ARAILKDVIE YVEEKYGIDIGFEVVRGDWS VRGVIDKLRN AILLKKRGYK IEKIDLDYYV LLDFF VAPNREEREY IMMKYNLSPDFIPLVLRQLI KIMANAMYGY YARKAGIVVI KIDKDYSTVL ELAKEVQLRV YEVDLDDLIIVGKGTTIGYV ERQVIPAALR 53 Sso7d SNS-dsDBD amino acid sequence ofSulfolobus solfataricus (see US 6,627,424) ATVKFKYKGE GRGAVSEKDAEKEVDISKIK PKELLQMLEK KVWRVGKMIS QKK FTYDEGGGKT 54 Sac7d SNS-dsDBD aminoacid sequence of Sulfolobus acidocaldarius VKVKFKYKGE RGAVSEKDAPEKEVDTSKIK KELLDMLARA KVWRVGKMVS EREKK FTYDDNGKTG 55 Pyrobaculumaerophilum Pae3192 amino acid sequence SKKQKLKFYD SPYTGIKVYR IKAKQAFETDLLGKKK QYEVIEKQTA RGPMMFAVAK 56 Pyrobaculum aerophilum Pae0384 aminoacid sequence AKQKLKFYDI PYTGIKVYRL KAKQSFETDK LGKKK YEVIEKETARGPMLFAVATS 57 Aeropyrum pernix Ape3192 amino acid sequence PKKEKIKFFDSPYTGKIFYR LVAKKYYETD VLGKA NYEVEIKETK RGKFRFAKAK 58 HMfA HMf familyarchaeal histone amino acid sequence of Methanothermus fervidusGELPIAPIGR SEAVKLAKHA IIKNAGAERV GRKTIKAED SDDARIALAK VLEEMGEEIA 59 HMfBHMf family archaeal histone amino acid sequence of Methanothermusfervidus ELPIAPIGRI EAIKLARHAG IKDAGAERVS RKTIKAEDI DDARITLAKILEEMGRDIAS 60 HpyA1 HMf family archaeal histone amino acid sequence ofPyrococcus strain GB-3a GELPIAPVDR KKAVEFARHA LIRKAGAERV GRKTVKAEDSEEAAKILAE YLEEYAIEVS 61 HpyA2 HMf family archaeal histone amino acidsequence of Pyrococcus strain GB-3a AELPIAPVDR RKAVDLAKHA LIRKAGAQRVGRKTVKAED SEQAAKLLAE HLEEKALEIA 62 Sso7d sequence non-specificDNA-binding domain amino acid sequence ATVKFKYKGE GRGAVSEKDA EKEVDISKIKPKELLQMLEK KVWRVGKMIS QK FTYDEGGGKT 63 Pyrococcus 3′-5′ exonucleasedomain amino acid sequence EELKLLAFDI WKKIDLPYVE SFDLPYLAKR KGRIHFDLYHDEIAKAWETG ETLYHEGEEF VVSSEREMIK AEKLGIKLTI VIRRTINLPT EGLERVAKYSGKGPIIMISY RFLKIIREKD GRDGSEPKMQ YTLEAVYEAI MEDAKATYEL ADEEEAKVITPDIIITYNGD RIGDMTAVEV FGKPKEKVYA GKEF 64 PCR Primer nucleotide sequenceGAAGAGCCAAGGACAGGTAC 65 PCR Primer nucleotide sequenceCCTCCAAATCAAGCCTCTAC 66 PCR Primer nucleotide sequenceCAGTGCAGTGCTTGATAACAGG 67 PCR Primer nucleotide sequenceGTAGTGCGCGTTTGATTTCC 68 PCR Primer nucleotide sequenceCCTGCTCTGCCGCTTCACGC 69 PCR Primer nucleotide sequenceCGAACGTCGCGCAGAGAAACAGG 70 PCR Primer nucleotide sequenceCTGATGAGTTCGTGTCCGTACAACTGGCGTAATC 71 PCR Primer nucleotide sequenceGTGCACCATGCAACATGAATAACAGTGGGTTATC 72 PCR Primer nucleotide sequenceGGGCGTTTTCCGTAACACTG 73 PCR Primer nucleotide sequenceTGACCACATACAATCGCCGT 74 PCR Primer nucleotide sequenceCTCCACAGGGTGAGGTCTAAGTGATGACA 75 PCR Primer nucleotide sequenceCAATCTCAGGGCAAGTTAAGGGAATAGTG 80 Pfu GenBank WP_011011325.1 P36H R762X²amino acid sequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPMESYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPNIVYLDFRALY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TX²QVGLTSWL EGKPVIRLFK VKKITGERHGQDVPTIREKV GEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWEIQLSRLVGQP KPSEEEYQRR PSIIITHNVS PSLLGHLLEE ANSFYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LANYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKS; KENGKFKIEHKIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINLSGENLERVAK LWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFGDRTFRHYIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILLAESVTAWGRK GGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRYQK wherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X² is selected from Q and N. 81Pfu GenBank WP_011011325.1 P36H R762Q amino acid sequence MILDVDYITELLRDDSKIEE TVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYN MQRIGDMTAVAIFGKPKEKV ELGKEFLPME AYERNEVAPN IVYLDFRALY KFCKDIPGFI DYRQKAIKLLYIELVWKELE ALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAK YDPKKHKYDATQQVGLTSWL EGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPYGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKS KENGKFKIEH KIVRIVDVEK REHPAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAK LWDVSRSSTG LRESYTGGFVPDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLETLAIYEQITRP GYIVLRGDGP AVLRILEGFG DRTFRHYIYA VEKKFLGKPI EYDIPFAKRYEFGKGPIIMI IKRFLRIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRKKEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRK GGESEEIKKK TKKRYAVIDEILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQK 82 Pfu GenBankWP_011011325.1 P36H A408S R762X² amino acid sequence MILDVDYITELLRDDSKIEE TVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYN MQRIGDMTAVAIFGKPKEKV ELGKEFLPME EGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAFITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KENGKFKIEHKIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINLSGENLERVAK LWDVSRSSTG DRTFRHYIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMIIKRFLRIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK AYERNEVAPNIVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TX²QVGLTSWL KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKS; LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFGKEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRK GGESEEIKKK TKKRYAVIDEILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQK wherein X² is selected fromQ, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments,X² is selected from Q and N. 83 Pfu GenBank WP_011011325.1 P36H A408SR762Q amino acid sequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPMESYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPNIVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TQQVGLTSWL EGKPVIRLFK VKKITGERHGQDVPTIREKV GEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWEIQLSRLVGQP KPSEEEYQRR PSIIITHNVS PSLLGHLLEE ANSFYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LANYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKS KENGKFKIEHKIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINLSGENLERVAK LWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFGDRTFRHYIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILLAESVTAWGRK GGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRYQK 84 Pfu GenBank WP_011011325.1 P36H R762X² with DNA bindingdomain amino acid sequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPMESYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPNIVYLDFRALY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TX²QVGLTSWL KKVWRVGKMI KQKK; EGKPVIRLFKVKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDLYADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVS PSLLGHLLEE ANSFYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEK GVKIKPGMVI EYYIENQVLPNIKKSGTGGG SFTYDEGGGK KENGKFKIEH KIVRIVDVEK REHPAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAK LWDVSRSSTG LRESYTGGFVPDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLETLAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKG TGRGAVSEKD DRTFRHYIYAVEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRKGGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQKEEKEVDISKI APKELLQMLE wherein X² is selected from Q, N, H, S, T, Y, C,M, W, A, I, L, F, V, P, and G; in some embodiments, X² is selected fromQ and N. 85 Pfu GenBank WP_011011325.1 P36H R762Q with DNA bindingdomain amino acid sequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPMEEGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF KENGKFKIEH KIVRIVDVEKREHPAVVDIF DIETLYHEGE DRTFRHYIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMISYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPNIVYLDFRALY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TQQVGLTSWL KKVWRVGKMI KQKK ITWKNIDLPYGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK VEVVSSEREM KRAEKLGIKLYHVITRTINL SGENLERVAK LWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKEAKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGPAVLRILEGFG GATVKFKYKG TGRGAVSEKD IKRFLRIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRKGGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQKEEKEVDISKI APKELLQMLE 86 Pfu GenBank WP_011011325.1 P36H A408S R762X²with DNA binding domain amino acid sequence MILDVDYITE LLRDDSKIEETVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKVELGKEFLPME AYERNEVAPN IVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELEALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TX²QVGLTSWLKKVWRVGKMI KQKK; EGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPYGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGKFKIEH KIVRIVDVEKREHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAKLWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKGTGRGAVSEKD DRTFRHYIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGHTQDPIEKILL AESVTAWGRK GGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPHISNRAILAEE YRKEDLRYQK EEKEVDISKI APKELLQMLE wherein X² is selected fromQ, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments,X² is selected from Q and N. 87 Pfu GenBank WP_011011325.1 P36H A408SR762Q with DNA binding domain amino acid sequence MILDVDYITE LLRDDSKIEETVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKVELGKEFLPME AYERNEVAPN IVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELEALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TQQVGLTSWLEGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPY GDSFDFPYLAEVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVS PSLLGHLLEEANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEK GVKIKPGMVIEYYIENQVLP NIKKSGTGGG KENGKFKIEH KIVRIVDVEK REHPAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAK LWDVSRSSTG LRESYTGGFVPDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLETLAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKG DRTFRHYIYA VEKKFLGKPIEYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATYNLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRK GGESEEIKKKTKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQK EEKEVDISKIKKVWRVGKMI KQKK SFTYDEGGGK TGRGAVSEKD APKELLQMLE 88 Pyrococcus DNApolymerase sequence including exonuclease domain and catalytic domain,P36H K762X² MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKVKDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALYKFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQKVAVAKRLAAK YDPRKHKYDA TX²QTGL; EGKPVIRLFK VKKITAERHG QDVPTIREKIGDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEH KIVRIVDAEKREHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAKLWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG DRTFRHYIYAVEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGREGGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQKwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 89 PyrococcusDNA polymerase sequence including exonuclease domain and catalyticdomain, P36H A408S K762X² MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPMESYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPNIVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLEVRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TX²QTGL; EGKPVIRLFK VKKITAERHGQDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEHKIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINLTGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFGDRTFRHYIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIMLAESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRWQK wherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X² is selected from Q and N. 90Pyrococcus DNA polymerase sequence including exonuclease domain andcatalytic domain, P36H K762Q MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPMESYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPNIVSLDFRALY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKPVIRLFKVKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDLYADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY KENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFVPDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFV DRTFRHYIYAVEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGREGGKSEEIKKK TKKKYALIDE EGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TQQTGLIVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP KETQARVLEA LAIYEQITRPGYIVLRGDGP AVLRILEGFG ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK 91Pyrococcus DNA polymerase sequence including exonuclease domain andcatalytic domain, P36H A408S K762Q MILDADYITE LLKDDSKIEE TVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPMEAYERNELAPN IVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINAEGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TQQTGL EGKPVIRLFK VKKITAERHGQDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEHKIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINLTGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFGDRTFRHYIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIMLAESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRWQK 92 Pyrococcus DNA polymerase sequence including exonucleasedomain and DNA binding domain, P36H K762X² MILDADYITE LLKDDSKIEETVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKVELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFI DYRQRAIKIL YIEFVWKELEALEFVDYINA EGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TX²QTGLTSWLKKVWRVGKMI KQKK; EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPYGDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVSPSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGEFKIEH KIVRIVDAEKREHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAKLWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKGTGRGAVSEKD DRTFRHYIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGHSQDPIEKIML AESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISNRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X² is selected fromQ, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments,X² is selected from Q and N. 93 Pyrococcus DNA polymerase sequenceincluding exonuclease domain and DNA binding domain, P36H K762QMILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYNMQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFIDYRQRAIKIL EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPYGDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVSPSLLKRLLDE ANSYYGYYGY KENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFVPDTLNREGCR RQKIKTKMKA AKARWYCKEC DRTFRHYIYA VEKKFLGRPI EYDIPFAKRYEFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRKKEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGRE YIEFVWKELE ALEFVDYINAEGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TQQTGLTSWL KKVWRVGKMI KQKKEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLPNIKKSGTGGG SFTYDEGGGK DTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRPGYIVLRGDGP AVLRILEGFG GATVKFKYKG TGRGAVSEKD GGKSEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLE 94Pyrococcus DNA polymerase sequence including exonuclease domain and DNAbinding domain, P36H A408S K762X² MILDADYITE LLKDDSKIEE TVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPMEAYERNELAPN IVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINAEGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TX²QTGLTSWL KKVWRVGKMI KQKK;EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLAEVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDEANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVIEYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGEFKIEH KIVRIVDAEK REHSAVVDIFDIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTGLRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFVKETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKG TGRGAVSEKDDRTFRHYIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIMLAESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X² is selected from Q, N, H, S,T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X² isselected from Q and N. 95 Pyrococcus DNA polymerase sequence includingexonuclease domain and sequence non-specific DNA binding domain, P36HA408S K762Q MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKVKDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRSLYKFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQKVAVAKRLAAK YDPRKHKYDA TQQTGLTSWL KKVWRVGKMI KQKK EGKPVIRLFK VKKITAERHGQDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKSGTGGGSFTYDEGGGK KENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREMKRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCRRQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRPGYIVLRGDGP AVLRILEGFG GATVKFKYKG TGRGAVSEKD DRTFRHYIYA VEKKFLGRPIEYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATYNLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKKTKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK EEKEVDISKIAPKELLQMLE 96 P36H K762X² variant of Deep Vent DNA polymerase amino acidsequence MILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPME SYADEEEAKVKDPDVIITYN MQRLGDMTAV DGKPIIRIFK VRKITAERHG QDVPAIRDKI GDEELKLLAFITWKKIDLPY GDSFDLPYLV EIKGRIHFDL KENGEFKVEY KIVRIIDAEK REHSAVIDIFDIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL DRNFRHYIYA VRKKFLGRPIEYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE AIFGKPKEKVELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELEALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TX²QTGLTAWLYAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKK;TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKECDTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFGYSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGREGAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQKwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 97 P36H K762Qvariant of Deep Vent DNA polymerase amino acid sequence MILDADYITELLKDDSQIDE EVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAVAIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKILYIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDATQQTGLTAWL DGKPIIRIFK VRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPYGDSFDLPYLV EIKGRIHFDL YAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVSPSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEKGVKVRPGMVI EYYIENQVLP NIKKK KENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYVPDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEALVIYEQITRP GYIVLRGDGP AVLRILEAFG DRNFRHYIYA VRKKFLGRPI EYDIPFAKRYEFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRKKEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK 98 P36H K762X² variant ofDeep Vent DNA polymerase amino acid sequence with DNA binding domainMILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDVIITYNMQRLGDMTAV AIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFIDYRQRAIKIL YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAARFDLRKHKYDA TX²QTGLTAWL KKVWRVGKMI KQKK; DGKPIIRIFK VRKITAERHG QDVPAIRDKIGDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKKGTGGG SFTYDEGGGKKENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG GATVKFKYKG TGRGAVSEKD DRNFRHYIYA VRKKFLGRPI EYDIPFAKRYEFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRKKEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLEwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 99 P36H K762Qvariant of Deep Vent DNA polymerase amino MILDADYITE LLKDDSQIDEDGKPIIRIFK VRKITAERHG KENGEFKVEY KIVRIIDAEK DRNFRHYIYA VRKKFLGRPI acidsequence with DNA binding domain EVWRLYFEHP LIDKGLIPME SYADEEEAKVKDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLYKFCKDFPGFI DYRQRAIKIL YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEKVAVAKRLAAR FDLRKHKYDA TQQTGLTAWL KKVWRVGKMI KQKK QDVPAIRDKI GDEELKLLAFITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWE AQLSRLVGQP KPDEREYERRPSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKKGTGGG SFTYDEGGGK REHSAVIDIFDIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAK LWDVSRSSTGLRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIP EGFYVRGFFVKETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKG TGRGAVSEKDEYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTYNLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKKTKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK EEKEVDISKIAPKELLQMLE 100 P36H K762X² K775S variant of Deep Vent DNA polymeraseamino acid sequence with sequence non-specific DNA binding domainMILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDVIITYNMQRLGDMTAV AIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFIDYRQRAIKIL YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAARFDLRKHKYDA TX²QTGLTAWL KKVWRVGKMI KQKK; DGKPIIRIFK VRKITAERHG QDVPAIRDKIGDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGKKENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG GATVKFKYKG TGRGAVSEKD DRNFRHYIYA VRKKFLGRPI EYDIPFAKRYEFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRKKEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLEwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 101 P36H K762QK775S variant of Deep Vent DNA polymerase amino acid sequence withsequence non-specific DNA binding domain MILDADYITE LLKDDSQIDEEVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKVELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELEALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAAR DGKPIIRIFK VRKITAERHGQDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI KENGEFKVEY KIVRIIDAEKREHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAKLWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIPEGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGP DRNFRHYIYA VRKKFLGRPIEYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTYNLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKKTKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEE FDLRKHKYDA TQQTGLTAWLKKVWRVGKMI KQKK EYYIENQVLP NIKKSGTGGG SFTYDEGGGK AVLRILEAFG GATVKFKYKGTGRGAVSEKD YRKEDLRWQK EEKEVDISKI APKELLQMLE 102 P36H K764X² variant ofThermococcus litoralis DNA polymerase MILDTDYITK LLKDDSAIEE EVWKLIFEHPLIDKGLIPME SYADEEEARV KDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFPRVAYARNELA ENIIYLDFRS GYRFCKDFPG MLDYRQRAIK RHYIEMTIRE KKAKEFLNYIDEEGRITTRG KAVEVVRDVV PHVAIAKRLA TEYDPRKHKY QSSX²QTGLDA DGKPIIRIFKIKAIKGERHG QDVPAMRGKI GDEELKLLAF ITWKNIDLPY GDNFDLPYLI AVEIKGRIHFKLGAEEIAAI MEAELAKLIG PNKPDEEEYK LYPSIIVTHN FIPSILGDLI LLANSYYGYMIEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR;KENGEFKIEL KTVRVLDAVK REHPAVVDIY DIETFYHEGD VDVVSNEREM KRAEKLGVRLDLFPVVRRTI WETEESMKKL QSVWDVSRSS RRLRTTYLGG VSPDTLEKEG AMRQDIKKKMGYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGSLPAVLRILEA DPHFQHYIYA VRKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKEVLGRDKEHPE NLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLW CKNYDVAPIVKSTIDPIEKK ECAESVTAWG IPGEKPELIK FVTKKRYAVI EAILKEGSVE RDLKDYKAIGGKISDRVILL FGYRKEDLRY wherein X² is selected from Q, N, H, S, T, Y, C,M, W, A, I, L, F, V, P, and G; in some embodiments, X² is selected fromQ and N. 103 P36H K764Q variant of Thermococcus litoralis DNA polymeraseMILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARV KDPDVIITYNPKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYARNELA ENIIYLDFRS GYRFCKDFPGMLDYRQRAIK RHYIEMTIRE KKAKEFLNYI DEEGRITTRG KAVEVVRDVV PHVAIAKRLATEYDPRKHKY QSSQQTGLDA DGKPIIRIFK IKAIKGERHG QDVPAMRGKI GDEELKLLAFITWKNIDLPY GDNFDLPYLI AVEIKGRIHF KLGAEEIAAI MEAELAKLIG PNKPDEEEYKLYPSIIVTHN FIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSEEKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR KENGEFKIEL KTVRVLDAVK REHPAVVDIYDIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSSRRLRTTYLGG VSPDTLEKEG AMRQDIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGFIAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA DPHFQHYIYA VRKKFLGREVEYDIPFAKRY EFGKGEIIMI IKRFVQVVKE VLGRDKEHPE NLPTYTLEAV AQYSMEDARATGNLVEWYLL YVKEPEKGLW CKNYDVAPIV KSTIDPIEKK ECAESVTAWG IPGEKPELIKFVTKKRYAVI EAILKEGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRY 104 P36H K764X²variant of Thermococcus litoralis DNA polymerase, sequence 2 (acc.ADK47977.1) MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARVKDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYERNELA ENIIYLDFRSSYRFCKDFPG MLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRG DGKPIIRIFKIKAIKGERHG QDVPAMRDKI GDEELKLLAF ITWKNIDLPY GDNFDLPYLI AVEIKGRIHFKLGAEEIAAI MEAELAKLIG PNKPDEEEYK LYPSIIVTHN FIPSILGDLI LLANSYYGYMIEEKFGFKVL NSKLPGLLEL LEVVRRDWSE KENGEFKIEL KSVRVVDAVK KEHPAVIDIYDIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSSRRLRTTYLGG VSPDTLEKEG AMRQEIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGFIAKETQAKVL DPHFQHYIYA VKKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKEVLGRDKENPE NLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLW CENYDIAPIVKATIDPVERK ECAESVTAWG ISGEKPEIIK FVTKKRYAVI EAILKDGSVE KAVEIVRDVLPHVAIAKRLA TEYDPEKHKY QSSX²QTGLDA EKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR;EKLVIHEQIT IISYIVLKGS LPAVLRILEA RDLKDYKAIG GKISDRVILL FGYRKEDLRYwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 105 P36H K764Qvariant of Thermococcus litoralis DNA polymerase, sequence 2 (acc.ADK47977.1) MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARVKDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYERNELA ENIIYLDFRSSYRFCKDFPG MLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRG KAVEIVRDVLPHVAIAKRLA TEYDPEKHKY QSSQQTGLDA DGKPIIRIFK IKAIKGERHG QDVPAMRDKIGDEELKLLAF ITWKNIDLPY GDNFDLPYLI AVEIKGRIHF KLGAEEIAAI MEAELAKLIGPNKPDEEEYK LYPSIIVTHN FIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLELLEVVRRDWSE EKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR KENGEFKIEL KSVRVVDAVKKEHPAVIDIY DIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTI WETEESMKKLQSVWDVSRSS RRLRTTYLGG VSPDTLEKEG AMRQEIKKKM GYPKARWYSK YADTDGFYATEYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA DPHFQHYIYAVKKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKE VLGRDKENPE NLPTYTLEAVAQYSMEDARA TGNLVEWYLL YVKEPEKGLW CENYDIAPIV KATIDPVERK ECAESVTAWGISGEKPEIIK FVTKKRYAVI EAILKDGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRY 106P36H R761X² variant of Thermococcus gorgonarius DNA polymeraseMILDTDYITE LLKDDSAIED EVWKLYFTHP LIDKGLIPME SYADEEGARV KDPDVLITYNIQRMGDRFAV AIFGQPKEKV ELGKEFFPME AYERNELAPN VYLDFRSLYP FCKDFPGFIPYRQRAIKILA IETTIREIEE KEFLDYINAK DKITTRGLEI RIVKEVTEKL AVAKRLAARGDPAKHKYDAE X²QVGLGAWLK DGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAFITWKNIDLPY GDNFDFAYLK EVKGRIHFDL YAEEIAQAWE AQLSRLVGQS KPDERELARRSIIITHNVSP SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAKSKYEVPPEKL IKIRPGTVIS YYIENQVLPA PKT; KENGEFKIDY TTVRVVRAEK KEHPAVVDIYDIETLYHEGE VDVVSTEKEM KRSEKLGVKF YPVIRRTINL TGEGLERVAR LWDVSRSSTGRESYAGGYVK DTLNREGCEE QKVKKKMKAT KARWYCKECA TDGFFATIPG GFYKRGFFVTETQARVLEAI VIYEQITRDL YIVLKGSGRI VERILRAFGY DRNFEHYIYA VKKKFLGRPIEYDIPFAKRY EFAEGPILMI IKRFLKVVKE ILGREGSEPK PTYTLEAVYE YSMEDAKVTYNLVEWFLLRK EPERGLWENI YDVAPQVGHK IDPIEKKLLD ESVTAWGRQY ADAETVKKKAKKKYAVIDEE LKHGDVEEAV KDYKATGPHV GDRAIPFDEF RKEDLRYQKT wherein X² isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 107 P36H R761Q variant ofThermococcus gorgonarius DNA polymerase MILDTDYITE LLKDDSAIED EVWKLYFTHPLIDKGLIPME SYADEEGARV KDPDVLITYN IQRMGDRFAV AIFGQPKEKV ELGKEFFPMEAYERNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILA DGKPVIRIFK VKKITAERHGQDVPAIRDKI GDEELKMLAF ITWKNIDLPY GDNFDFAYLK EVKGRIHFDL YAEEIAQAWEAQLSRLVGQS KPDERELARR SIIITHNVSP SLLGDLLEER NSFYGYYGYA KENGEFKIDYTTVRVVRAEK KEHPAVVDIY DIETLYHEGE VDVVSTEKEM KRSEKLGVKF YPVIRRTINLTGEGLERVAR LWDVSRSSTG RESYAGGYVK DTLNREGCEE QKVKKKMKAT KARWYCKECADRNFEHYIYA VKKKFLGRPI EYDIPFAKRY EFAEGPILMI IKRFLKVVKE ILGREGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENI YDVAPQVGHK IDPIEKKLLDESVTAWGRQY IETTIREIEE KEFLDYINAK DKITTRGLEI RIVKEVTEKL AVAKRLAARGDPAKHKYDAE QQVGLGAWLK KFGFKVLYAD LPGLLELEYE VRRDWSEIAK SKYEVPPEKLIKIRPGTVIS YYIENQVLPA PKT TDGFFATIPG GFYKRGFFVT ETQARVLEAI VIYEQITRDLYIVLKGSGRI VERILRAFGY ADAETVKKKA KKKYAVIDEE LKHGDVEEAV KDYKATGPHVGDRAIPFDEF RKEDLRYQKT 108 P36H R761X² variant of Thermococcuskodakarensis DNA polymerase MILDTDYITE LLKDDSAIEE EVWKLYFTHP LIDKGLVPMESYADEEGARV KDPDVLITYN IQRMGDRFAV AVFGQPKEKV ELGKEFLPME AYERNELAPNVYLDFRSLYP FCKDFPGFIP YRQRAIKILA ITMTIKEIEE MEFLKYINAK GKITTRGLEIRIVKEVTEKL AVAKRLAARG DPTKHKYDAE X²QVGLSAWLK DGKPVIRIFK VKKITAERHGQDVPAIRDKI GDEELKMLAF ITWKNVDLPY GDNFDFAYLK EVKGRIHFDL YAEEITTAWEAQLSRLIGQS KPDEKELARR SIIITHNVSP SLLGDLLEER NSYYGYYGYA KYGFKVIYSDLPGALELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVIS YYIENQVLPA PKGT; KENGEFKIEYTVVTVKRVEK REHPAVIDIY DIETLYHEGE VDVVSTEREM KRCEKLGINF YPVIRRTINLTGENLERVAR LWDVSRSSTG RQSYEGGYVK DTLNREGCKE QKIKKKMKAT RARWYCKECATDGFFATIPG GFYKRGFFVT ETQARVLEAL VIHEQITRDL YIVLKGSGRI VERILRAFGYDRTFEHYFYA VQKKFLGRPV EYDIPFAKRY EFAEGPILMI IKRFLRVVKE ALGRDGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENI YDVAPQVGHR IDPIERKLLDESVTAWGREY ADAETVKKKA KKKYAVIDEE LKDGDVEKAV KDYKATGPHV GDRAIPFDEFRKEDLRYQKT wherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I,L, F, V, P, and G; in some embodiments, X² is selected from Q and N. 109P36H R761Q variant of Thermococcus kodakarensis DNA polymeraseMILDTDYITE LLKDDSAIEE EVWKLYFTHP LIDKGLVPME SYADEEGARV KDPDVLITYNIQRMGDRFAV AVFGQPKEKV ELGKEFLPME AYERNELAPN VYLDFRSLYP FCKDFPGFIPYRQRAIKILA ITMTIKEIEE MEFLKYINAK GKITTRGLEI RIVKEVTEKL AVAKRLAARGDPTKHKYDAE QQVGLSAWLK DGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAFITWKNVDLPY GDNFDFAYLK EVKGRIHFDL YAEEITTAWE AQLSRLIGQS KPDEKELARRSIIITHNVSP SLLGDLLEER NSYYGYYGYA KYGFKVIYSD LPGALELEYE VRRDWSEIAKSKYEVPPEKL VKIRPGTVIS YYIENQVLPA PKGT KENGEFKIEY TVVTVKRVEK REHPAVIDIYDIETLYHEGE VDVVSTEREM KRCEKLGINF YPVIRRTINL TGENLERVAR LWDVSRSSTGRQSYEGGYVK DTLNREGCKE QKIKKKMKAT RARWYCKECA TDGFFATIPG GFYKRGFFVTETQARVLEAL VIHEQITRDL YIVLKGSGRI VERILRAFGY DRTFEHYFYA VQKKFLGRPVEYDIPFAKRY EFAEGPILMI IKRFLRVVKE ALGRDGSEPK PTYTLEAVYE YSMEDAKVTYNLVEWFLLRK EPERGLWENI YDVAPQVGHR IDPIERKLLD ESVTAWGREY ADAETVKKKAKKKYAVIDEE LKDGDVEKAV KDYKATGPHV GDRAIPFDEF RKEDLRYQKT 110 P36H K761X²variant of Thermococcus species 9°N-7 DNA polymerase MILDTDYITELLKDDSAIED EVWKLYFNHP LIDKGLIPME SYADGSEARV KDPDVLITYN IQRMGDRFAVAVFGKPKEKV ELGREFFPME AYKRNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILANGKPVIRVFK VKKVTAKRHG QDVPAIRDRI GDEELTMLAF ITWKKIDLPY GDNFDFAYLKEVKGRIHFDL YAEEIAQAWE AQLSRLIGQS KPDERELARR SIIITHNVSP SLLGDLLEERNSFYGYYGYA KENGEFKIEY TVVKVKRAEK RAHPAVVDIY DIETLYHEGE VDVVSTEKEMKRCEELGIKF YPVIRRTINL SGEGLERVAR LWDVSRSSTG RGGYAGGYVK DTLNREGCKEQKIKRKMKAT KARWYCKECA DRTFEHYFYA VQKKFLGRPI EYDIPFAKRY EFGTGPILMIIKRFLRVVRE TLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWDNIYDVAPEVGHK VDPLEKKLLD ESVTAWGREY IEMVIRELEE KEFLKYINPK GKITTRGLEIRIVKEVTEKL AVAKRLAARG DPTKHRYDAE X²QVGLGAWLK KFGFKVLYAD LPGLLELEYEVRRDWSEIAK SKYEVPPEKL VKIRPGTVIS YYIENQVLPA VKGKK; TDGLHATIPG GFYVRGFFVTETQARVLEAI VIHEQITRDL YIVLKGSGRI VERILKAFGY ADAETVKKKA KKKYAVIDEELKHGDVEEAV RDYKATGPHV GDRAIPADEF RKEDLRYQKT wherein X² is selected fromQ, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments,X² is selected from Q and N. 111 P36H K761Q variant of Thermococcusspecies 9°N-7 DNA polymerase MILDTDYITE LLKDDSAIED EVWKLYFNHP LIDKGLIPMESYADGSEARV KDPDVLITYN IQRMGDRFAV AVFGKPKEKV ELGREFFPME AYKRNELAPNVYLDFRSLYP FCKDFPGFIP YRQRAIKILA IEMVIRELEE KEFLKYINPK GKITTRGLEIRIVKEVTEKL AVAKRLAARG DPTKHRYDAE QQVGLGAWLK NGKPVIRVFK VKKVTAKRHGQDVPAIRDRI GDEELTMLAF ITWKKIDLPY GDNFDFAYLK EVKGRIHFDL YAEEIAQAWEAQLSRLIGQS KPDERELARR SIIITHNVSP SLLGDLLEER NSFYGYYGYA KFGFKVLYADLPGLLELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVIS YYIENQVLPA VKGKK KENGEFKIEYTVVKVKRAEK RAHPAVVDIY DIETLYHEGE VDVVSTEKEM KRCEELGIKF YPVIRRTINLSGEGLERVAR LWDVSRSSTG RGGYAGGYVK DTLNREGCKE QKIKRKMKAT KARWYCKECATDGLHATIPG GFYVRGFFVT ETQARVLEAI VIHEQITRDL YIVLKGSGRI VERILKAFGYDRTFEHYFYA VQKKFLGRPI EYDIPFAKRY EFGTGPILMI IKRFLRVVRE TLGRDGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWDNI YDVAPEVGHK VDPLEKKLLDESVTAWGREY ADAETVKKKA KKKYAVIDEE LKHGDVEEAV RDYKATGPHV GDRAIPADEFRKEDLRYQKT 112 P40H E775Q variant of Pyrobaculum calidifontis DNApolymerase MRFWPLDATY YFYAKCDKCD FLKVVAKVPE IDKGVVPCAW PPLRVLAFDIFEAEGRDDRR SERAKALGVP IVDEFPEIKV NDPAKRPTLM PLDQVAAASV KGAIVLEPKPEPHEPDPPEG RAVREEAKKY VGARWYKKEV TDSLFVKKSG AKKRYAGLLR ILKSKSVGEALDKELDEYKA GPGKVSERAM VLGVKESDLK SVVGGVPEVR ASLAKSYLSR DVRKLREAALNVVEAREAGK EVYNERGSPD VIRGFVDFVK LRVDRLGGVP KTLDRVAEYF RYVLDDVRSTGNRVEWMLLR GLYSDVLVLD VVVAPEVGHR PPDSPEYRLL AESVTAFARA AVDRLVKYVEDGRIDIVGFE RERVVKYVRE YGPHVHAALE PYIFVDDASK TGRVQKSLLD VFGVDGEGRRVAPVEAVEVV GAPGVVDVYE LGPLPLYEVV PLRDPVVMLA EFDPDVIVGY QQSVYGHWSVGVMKRSERVL LGLAEKLLPF YAYRMGEVAP FSSMYPNIMM FRKAPTGFIP DERQRALKVMILLDVVEYAK ERHGIEIKVD VVRGDWCELA VVERLKAYKF LKRRGYKVGK VDVDYYIEKQ FLGVVLVDRRFRH ERRFFGRPTI ADIRYYMRYM EWAGVEEGFP VKTSDGREEV NSNGFDWPYLVGRANVDLYN IPGHKVYEYW LIQLSSVSGL NREEREYEPY KYNLSPDTYL AVLKHLVELRANAMYGYLGW RLGIEVIYGD KDYERVLFTE KEVQLNVVEL DLDDLIIWKT GTTVGYVIVRVIPAALRIAE 113 P42H E778Q variant of Pyrobaculum aerophilum DNApolymerase MKFKLWPLDA RHYFYADCPA RSFLKIVARV YMLDMGVVPC GFPPPLRVLAVEVFEASGRD PYLAERARAL LYNIVDEFPE EYWRDQGKRP SGLPLDQVAA EPYKGAIVLETYLERGEPDP ELRKRVREEL TYSVVGGVPE CDPESVRSQL PEDVRKLREA SWNTVDAEATFDIEVYNERG DRSVLRSFID GIPLKVDRVG IKLKTLDRVA LLRQYVIDDV ASVGNRVEWMPRPGLYSDVL PGGVYVAPEV KKYPPDSPEY VRIFGISESG GRVAPVEEVV AAALPGVSGVGEKLGNLPVY TPDPLRDPVI FVREFDPDVI GAPQQSVYGH EYFGVMKREE KSTYGLAEKLLLRYAYRLGE ALDFSSMYPN GHRFRREPPG RVLDERQRAL DRVVVVDRRF AVERRYLGRPYEADIRFYMR KVAEWGGVTE LLAVQASDGR VGYNSNQFDW WSVTGRANVD RVLVPGHKIYLPFLIQLSSV VAPNREEREY IMMKYNLSPD FIPLVLRQLI KIMANAMYGY TGWVGARWYKYGDTDSLFVK FTEAKKRYAG IELILTSRDV WKTLDKELDE VVKGGEKVSE IAEVIGIKEGKEVAESVTAF KSGDVEKLVK LLRDGRIDIV SEARQKVVKY YKAYPPHVHA RAVPYIFIDDDLKTGRSQRT ARAILKDVIE YVEEKYGIDI GFEVVRGDWS VRGVIDKLRN AILLKKRGYKIEKIDLDYYV LLDFF YARKAGIVVI KIDKDYSTVL ELAKEVQLRV YEVDLDDLII VGKGTTIGYVERQVIPAALR 114 Pyrococcus DNA polymerase sequence including exonucleasedomain and catalytic domain, P36H MILDADYITE LLKDDSKIEE TVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPMEAYERNELAPN IVSLDFRALY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINAEGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TKQTGL EGKPVIRLFK VKKITAERHGQDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEHKIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINLTGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFGDRTFRHYIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIMLAESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRWQK 115 Pyrococcus DNA polymerase N-terminal domain comprising auracil-binding pocket, P36H MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPMEEGKPVIRLFK VKKITAERHG QDVPTIREKI G KENGEFKIEH KIVRIVDAEK REHSAVVDIFDRTFRHYIYA VEKKFLGRPI EYDIPFAKRY 116 Pfu DNA polymerase (GenBank Acc.No. WP_011011325.1) N-terminal domain comprising a uracil-bindingpocket, P36H MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPME EGKPVIRLFKVKKITGERHG QDVPTIREKV G KENGKFKIEH KIVRIVDVEK REHPAVVDIF DRTFRHYIYAVEKKFLGKPI EYDIPFAKRY 117 Deep Vent DNA polymerase N-terminal domaincomprising a uracil-binding pocket, P36H MILDADYITE LLKDDSQIDEEVWRLYFEHP LIDKGLIPME DGKPIIRIFK VRKITAERHG QDVPAIRDKI G KENGEFKVEYKIVRIIDAEK REHSAVIDIF DRNFRHYIYA VRKKFLGRPI EYDIPFAKRY 118 Thermococcuslitoralis DNA polymerase N-terminal domain comprising a uracil-bindingpocket, P36H MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME DGKPIIRIFKIKAIKGERHG QDVPAMRGKI G KENGEFKIEL KTVRVLDAVK REHPAVVDIY DPHFQHYIYAVRKKFLGREV EYDIPFAKRY 119 Thermococcus gorgonarius DNA polymeraseN-terminal domain comprising a uracil-binding pocket, P36H MILDTDYITELLKDDSAIED EVWKLYFTHP LIDKGLIPME DGKPVIRIFK VKKITAERHG QDVPAIRDKI GKENGEFKIDY TTVRVVRAEK KEHPAVVDIY DRNFEHYIYA VKKKFLGRPI EYDIPFAKRY 120Thermococcus kodakarensis DNA polymerase N-terminal domain comprising auracil-binding pocket, P36H MILDTDYITE LLKDDSAIEE EVWKLYFTHP LIDKGLVPMEDGKPVIRIFK VKKITAERHG QDVPAIRDKI G KENGEFKIEY TVVTVKRVEK REHPAVIDIYDRTFEHYFYA VQKKFLGRPV EYDIPFAKRY 121 Thermococcus species 9°N-7 DNApolymerase N-terminal domain comprising a uracil-binding pocket, P36HMILDTDYITE LLKDDSAIED EVWKLYFNHP LIDKGLIPME NGKPVIRVFK VKKVTAKRHGQDVPAIRDRI G KENGEFKIEY TVVKVKRAEK RAHPAVVDIY DRTFEHYFYA VQKKFLGRPIEYDIPFAKRY 127 Pfu GenBank WP_011011325.1 P36X¹ R762X² amino acidsequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPME SYADENEAKVEGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPY KENGKFKIEHKIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM DRTFRX¹YIYA VEKKFLGKPIEYDIPFAKRY EFGKGPIIMI IKRFLRIIRE KDPDIIVTYN MQRIGDMTAV AIFGKPKEKVELGKEFLPME AYERNEVAPN IVYLDFRALY KFCKDIPGFI DYRQKAIKLL YIELVWKELEALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TX²QVGLTSWLGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKS; KRAEKLGIKL YHVITRTINL SGENLERVAK LWDVSRSSTGLRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIP EGFYKRGFFVKETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFG TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRKGGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQKwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H; and wherein X² is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, P, and G; in some embodiments, X² is selectedfrom Q and N. 128 Pfu GenBank WP_011011325.1 P36X¹ R762Q amino acidsequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPME SYADENEAKVKDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPN IVYLDFRALYKFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLE VRIVKEVIQKVAVAKKLAAK YDPKKHKYDA TQQVGLTSWL EGKPVIRLFK VKKITGERHG QDVPTIREKVGEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQPKPSEEEYQRR PSIIITHNVS PSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LANYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKS; KENGKFKIEH KIVRIVDVEKREHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAKLWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFG DRTFRX¹YIYAVEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRKGGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQKwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H. 129 Pfu GenBank WP_011011325.1 P36X¹ A408S R762X²amino acid sequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPMESYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPNIVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK EGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPYGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKKENGKFKIEH KIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVITRTINL SGENLERVAK LWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKEAKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP DRTFRX¹YIYAVEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRKGGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH VAVAKKLAAK YDPKKHKYDATX²QVGLTSWL GVKIKPGMVI EYYIENQVLP NIKKS; GYIVLRGDGP AVLRILEGFGISNRAILAEE YRKEDLRYQK wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H; and wherein X² is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X²is selected from Q and N. 130 Pfu GenBank WP_011011325.1 P36X¹ A408SR762Q amino acid sequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPMESYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPNIVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLEVRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TQQVGLTSWL EGKPVIRLFK VKKITGERHGQDVPTIREKV GEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWEIQLSRLVGQP KPSEEEYQRR PSIIITHNVS PSLLGHLLEE ANSFYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LANYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKS; KENGKFKIEHKIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINLSGENLERVAK LWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKECDTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFGDRTFRX¹YIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRE TIGRDGSEPKPTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILLAESVTAWGRK GGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEEYRKEDLRYQK wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 131 Pfu GenBank WP_011011325.1 P36X¹R762X² with DNA binding domain amino acid sequence MILDVDYITE LLRDDSKIEETVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKVELGKEFLPME AYERNEVAPN IVYLDFRALY KFCKDIPGFI DYRQKAIKLL YIELVWKELEALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TXQVGLTSWLKKVWRVGKMI KQKK; EGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPYGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGKFKIEH KIVRIVDVEKREHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAKLWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKGTGRGAVSEKD DRTFRX¹YIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGHTQDPIEKILL AESVTAWGRK GGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPHISNRAILAEE YRKEDLRYQK EEKEVDISKI APKELLQMLE wherein X¹ is any amino acidother than P; in some embodiments, X¹ is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H; and wherein X²is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 132 Pfu GenBankWP_011011325.1 P36X¹ R762Q with DNA binding domain amino acid sequenceMILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYNMQRIGDMTAV AIFGKPKEKV ELGKEFLPME AYERNEVAPN IVYLDFRALY KFCKDIPGFIDYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAKYDPKKHKYDA TQQVGLTSWL KKVWRVGKMI KQKK; EGKPVIRLFK VKKITGERHG QDVPTIREKVGEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQPKPSEEEYQRR PSIIITHNVS PSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LANYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGKKENGKFKIEH KIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVITRTINL SGENLERVAK LWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKEAKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGPAVLRILEGFG GATVKFKYKG TGRGAVSEKD DRTFRX¹YIYA VEKKFLGKPI EYDIPFAKRYEFGKGPIIMI IKRFLRIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRKKEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRK GGESEEIKKK TKKRYAVIDEILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQK EEKEVDISKI APKELLQMLEwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H. 133 Pfu GenBank WP_011011325.1 P36X¹ A408S R762X²with DNA binding domain amino acid sequence MILDVDYITE LLRDDSKIEETVWKLYLEHP LIDKGLIPME SYADENEAKV KDPDIIVTYN MQRIGDMTAV AIFGKPKEKVELGKEFLPME AYERNEVAPN IVYLDFRSLY KFCKDIPGFI DYRQKAIKLL YIELVWKELEALEFVKYINS EGKVITRGLE VRIVKEVIQK VAVAKKLAAK YDPKKHKYDA TXQVGLTSWLKKVWRVGKMI KQKK; EGKPVIRLFK VKKITGERHG QDVPTIREKV GEEELKILAF ITWKNIDLPYGDSFDFPYLA EVKGRIHFDL YADEIAKAWE IQLSRLVGQP KPSEEEYQRR PSIIITHNVSPSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LANYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGKFKIEH KIVRIVDVEKREHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINL SGENLERVAKLWDVSRSSTG LRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKGTGRGAVSEKD DRTFRX¹YIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDIAPQVGHTQDPIEKILL AESVTAWGRK GGESEEIKKK TKKRYAVIDE ILKHGDVEEA LHEYKAIGPHISNRAILAEE YRKEDLRYQK EEKEVDISKI APKELLQMLE wherein X¹ is any amino acidother than P; in some embodiments, X¹ is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H; and wherein X²is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 134 Pfu GenBankWP_011011325.1 P36X¹ A408S R762Q with DNA binding domain amino acidsequence MILDVDYITE LLRDDSKIEE TVWKLYLEHP LIDKGLIPME SYADENEAKVKDPDIIVTYN MQRIGDMTAV AIFGKPKEKV EGKPVIRLFK VKKITGERHG QDVPTIREKVGEEELKILAF ITWKNIDLPY GDSFDFPYLA EVKGRIHFDL YADEIAKAWE KENGKFKIEHKIVRIVDVEK REHPAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVITRTINLSGENLERVAK DRTFRX¹YIYA VEKKFLGKPI EYDIPFAKRY EFGKGPIIMI IKRFLRIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY ELGKEFLPME AYERNEVAPN IVYLDFRSLYKFCKDIPGFI DYRQKAIKLL YIELVWKELE ALEFVKYINS EGKVITRGLE VRIVKEVIQKVAVAKKLAAK YDPKKHKYDA TQQVGLTSWL KKVWRVGKMI KQKK; IQLSRLVGQP KPSEEEYQRRPSIIITHNVS PSLLGHLLEE ANSFYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIALANYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK LWDVSRSSTGLRESYTGGFV PDTLNLEGCK RQKIKTKMKE AKARWYCKEC DTDGLYATIP EGFYKRGFFVKETQARVLET LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKG TGRGAVSEKDNLVEWFLLRK KEPEKGLWEN NYDIAPQVGH TQDPIEKILL AESVTAWGRK GGESEEIKKKTKKRYAVIDE ILKHGDVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRYQK EEKEVDISKIAPKELLQMLE wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 135 Pyrococcus DNA polymerasesequence including exonuclease domain and catalytic domain, P36X¹ K762X²MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYNMQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFIDYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQK VAVAKRLAAKYDPRKHKYDA TXQTGL; EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAFITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERRPSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEH KIVRIVDAEK REHSAVVDIFDIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTGLRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFVKETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG DRTFRX¹YIYA VEKKFLGRPIEYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATYNLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKKTKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK wherein X¹ is anyamino acid other than P; in some embodiments, X¹ is selected from Q, N,H, S, T, Y, C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H; andwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 136 PyrococcusDNA polymerase sequence including exonuclease domain and catalyticdomain, P36X¹ A408S K762X² MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPMESYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPNIVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLEVRIVKEVTQK VAVAKRLAAK EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAFITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERRPSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKIKPGMVI KENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFVPDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLEALAIYEQITRP GYIVLRGDGP DRTFRX¹YIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMIIKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWENNYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEALHEYKAIGPH ISNRAILAEE YDPRKHKYDA TXQTGL; EYYIENQVLP AVLRILEGFGYRKEDLRWQK wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H; and wherein X² is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X²is selected from Q and N. 137 Pyrococcus DNA polymerase sequenceincluding exonuclease domain and catalytic domain, P36X¹ K762QMILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYNMQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFIDYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQK VAVAKRLAAKYDPRKHKYDA TQQTGL; EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAFITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERRPSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEH KIVRIVDAEK REHSAVVDIFDIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTGLRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFVKETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG DRTFRX¹YIYA VEKKFLGRPIEYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATYNLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKKTKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK wherein X¹ is anyamino acid other than P; in some embodiments, X¹ is selected from Q, N,H, S, T, Y, C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H. 138Pyrococcus DNA polymerase sequence including exonuclease domain andcatalytic domain, P36X¹ A408S K762Q MILDADYITE LLKDDSKIEE TVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPMEAYERNELAPN IVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINAEGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TQQTGL; EGKPVIRLFKVKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDLYADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLPKENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKAAKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGPAVLRILEGFG DRTFRX¹YIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGHSQDPIEKIML AESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISNRAILAEE YRKEDLRWQK wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 139 Pyrococcus DNA polymerasesequence including exonuclease domain and DNA binding domain, P36X¹K762X² MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYNEGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLAKENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLDRTFRX¹YIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPKMQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFIDYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQK VAVAKRLAAKYDPRKHKYDA TXQTGLTSWL KKVWRVGKMI KQKK; EVKGRIHFDL YADEIAKAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGKYHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKAAKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGPAVLRILEGFG GATVKFKYKG TGRGAVSEKD PTYTLEAVYE YSMEDAKATY NLVEWFLLRKKEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLEwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H; wherein X² is selected from Q, N, H, S, T, Y, C,M, W, A, I, L, F, V, P, and G; in some embodiments, X² is selected fromQ and N. 140 Pyrococcus DNA polymerase sequence including exonucleasedomain and DNA binding domain, P36X¹ K762Q MILDADYITE LLKDDSKIEETVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKVELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFI DYRQRAIKIL YIEFVWKELEALEFVDYINA EGKIITRGLE VRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TQQTGLTSWLKKVWRVGKMI KQKK; EGKPVIRLFK VKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPYGDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVSPSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEKGVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGEFKIEH KIVRIVDAEKREHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAKLWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKGTGRGAVSEKD DRTFRX¹YIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRETIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGHSQDPIEKIML AESVTAWGRE GGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISNRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X¹ is any amino acidother than P; in some embodiments, X¹ is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H. 141 PyrococcusDNA polymerase sequence including exonuclease domain and DNA bindingdomain, P36X¹ A408S K762X² MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPMESYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPNIVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE EGKPVIRLFK VKKITAERHGQDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKAAKARWYCKEC DTDGLYATIP DRTFRX¹YIYA VEKKFLGRPI EYDIPFAKRY EFGKGPIIMIIKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRK KEPEKGLWENNYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKK ALEFVDYINA EGKIITRGLEVRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TXQTGLTSWL KKVWRVGKMI KQKK; KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGKEGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKGTGRGAVSEKD TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQKEEKEVDISKI APKELLQMLE wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H; and wherein X² is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X²is selected from Q and N. 142 Pyrococcus DNA polymerase sequenceincluding exonuclease domain and sequence non-specific DNA bindingdomain, P36X¹ A408S K762Q MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPMESYADEEEAKV KDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPNIVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLEVRIVKEVTQK VAVAKRLAAK YDPRKHKYDA TQQTGLTSWL KKVWRVGKMI KQKK; EGKPVIRLFKVKKITAERHG QDVPTIREKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDLYADEIAKAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLPNIKKSGTGGG SFTYDEGGGK KENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGEVEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFVPDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLEALAIYEQITRP GYIVLRGDGP AVLRILEGFG GATVKFKYKG TGRGAVSEKD DRTFRX¹YIYAVEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGREGGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQKEEKEVDISKI APKELLQMLE wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 143 P36X¹ K762X² variant of DeepVent DNA polymerase amino acid sequence MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPMEAYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELE ALEFVDYINAEGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TXQTGLTAWL DGKPIIRIFKVRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDLYAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKK;KENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG DRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIREPLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGHSKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISKRAILAEE YRKEDLRWQK wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H; and wherein X² is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X²is selected from Q and N. 144 P36X¹ K762Q variant of Deep Vent DNApolymerase amino acid sequence MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPMEAYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELE ALEFVDYINAEGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TQQTGLTAWL DGKPIIRIFKVRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDLYAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGYEKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKK;KENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG DRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIREPLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGHSKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPHISKRAILAEE YRKEDLRWQK wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 145 P36X¹ K762X² variant of DeepVent DNA polymerase amino acid sequence with DNA binding domainMILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDVIITYNMQRLGDMTAV AIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFIDYRQRAIKIL YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAARFDLRKHKYDA TXQTGLTAWL KKVWRVGKMI KQKK; DGKPIIRIFK VRKITAERHG QDVPAIRDKIGDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKKGTGGG SFTYDEGGGKKENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKAAKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGPAVLRILEAFG GATVKFKYKG TGRGAVSEKD DRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRYEFAKGPIIMI IKRFLKVIRE PLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWYLLRKKEPEKGLWEG EYDVAPEVGH SKDPIEKKML AESVTAWGRE GAKPEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISKRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLEwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H; and wherein X² is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, P, and G; in some embodiments, X² is selectedfrom Q and N. 146 P36X¹ K762Q variant of Deep Vent DNA polymerase aminoacid sequence with DNA binding domain MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV DGKPIIRIFK VRKITAERHG QDVPAIRDKI GDEELKLLAFITWKKIDLPY KENGEFKVEY KIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREMDRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIRE KDPDVIITYNMQRLGDMTAV AIFGKPKEKV ELGREFFPME AYERNELAPN LVSLDFRSLY KFCKDFPGFIDYRQRAIKIL YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAARFDLRKHKYDA TQQTGLTAWL KKVWRVGKMI KQKK; GDSFDLPYLV EIKGRIHFDL YAHEIAEAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYIKLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKKGTGGGSFTYDEGGGK KRAEKLGIKL YHVIRRTINL TGKGLERVAK LWDVSRSSTG LRESYAGGYVPDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIP EGFYVRGFFV KETQAKVLEALVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKG TGRGAVSEKD PLGRDGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKMLAESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEEYRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H. 147 P36X¹ K762X² K775Svariant of Deep Vent DNA polymerase amino acid sequence with sequencenon-specific DNA binding domain MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPMEAYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL YIEFVRKELE ALEFVDYINAEGKIITRGLE VKIVKEVTEK VAVAKRLAAR FDLRKHKYDA TXQTGLTAWL KKVWRVGKMI KQKK;DGKPIIRIFK VRKITAERHG QDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLVEIKGRIHFDL YAHEIAEAWE AQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDEANSYYGYYGY EKFGFKVLYI KLPGLLELEY IVRRDWSEIA LSKYEIPPEK GVKVRPGMVIEYYIENQVLP NIKKSGTGGG SFTYDEGGGK KENGEFKVEY KIVRIIDAEK REHSAVIDIFDIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGKGLERVAK LWDVSRSSTGLRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKEC DTDGLYATIP EGFYVRGFFVKETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKG TGRGAVSEKDDRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKMLAESVTAWGRE GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEEYRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H; and wherein X² isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 148 P36X¹ K762Q K775Svariant of Deep Vent DNA polymerase amino acid sequence with sequencenon-specific DNA binding domain MILDADYITE LLKDDSQIDE EVWRLYFEHPLIDKGLIPME SYADEEEAKV KDPDVIITYN MQRLGDMTAV AIFGKPKEKV ELGREFFPMEAYERNELAPN LVSLDFRSLY KFCKDFPGFI DYRQRAIKIL DGKPIIRIFK VRKITAERHGQDVPAIRDKI GDEELKLLAF ITWKKIDLPY GDSFDLPYLV EIKGRIHFDL YAHEIAEAWEAQLSRLVGQP KPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY KENGEFKVEYKIVRIIDAEK REHSAVIDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINLTGKGLERVAK LWDVSRSSTG LRESYAGGYV PDTLNREGCR RQEIKRKMKA AKARWYCKECDRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRY EFAKGPIIMI IKRFLKVIRE PLGRDGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWYLLRK KEPEKGLWEG EYDVAPEVGH SKDPIEKKMLAESVTAWGRE YIEFVRKELE ALEFVDYINA EGKIITRGLE VKIVKEVTEK VAVAKRLAARFDLRKHKYDA TQQTGLTAWL KKVWRVGKMI KQKK; EKFGFKVLYI KLPGLLELEY IVRRDWSEIALSKYEIPPEK GVKVRPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGK DTDGLYATIPEGFYVRGFFV KETQAKVLEA LVIYEQITRP GYIVLRGDGP AVLRILEAFG GATVKFKYKGTGRGAVSEKD GAKPEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISKRAILAEEYRKEDLRWQK EEKEVDISKI APKELLQMLE wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H. 149 P36X¹ K764X² variantof Thermococcus litoralis DNA polymerase MILDTDYITK LLKDDSAIEEEVWKLIFEHP LIDKGLIPME SYADEEEARV KDPDVIITYN PKIQRMGDSF YEAVLGKTKSTYELGKEFFP RVAYARNELA ENIIYLDFRS GYRFCKDFPG MLDYRQRAIK RHYIEMTIREKKAKEFLNYI DEEGRITTRG KAVEVVRDVV PHVAIAKRLA TEYDPRKHKY QSSXQTGLDADGKPIIRIFK IKAIKGERHG QDVPAMRGKI GDEELKLLAF ITWKNIDLPY GDNFDLPYLIAVEIKGRIHF KLGAEEIAAI MEAELAKLIG PNKPDEEEYK LYPSIIVTHN FIPSILGDLILLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGTDPDYYIENQV WLKR; KENGEFKIEL KTVRVLDAVK REHPAVVDIY DIETFYHEGD VDVVSNEREMKRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSS RRLRTTYLGG VSPDTLEKEGAMRQDIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQITIISYIVLKGS LPAVLRILEA DPHFQX¹YIYA VRKKFLGREV EYDIPFAKRY EFGKGEIIMIIKRFVQVVKE VLGRDKEHPE NLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLWCKNYDVAPIV KSTIDPIEKK ECAESVTAWG IPGEKPELIK FVTKKRYAVI EAILKEGSVERDLKDYKAIG GKISDRVILL FGYRKEDLRY wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H; and wherein X² isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 150 P36X¹ K764Q variantof Thermococcus litoralis DNA polymerase MILDTDYITK LLKDDSAIEEEVWKLIFEHP LIDKGLIPME SYADEEEARV KDPDVIITYN PKIQRMGDSF YEAVLGKTKSTYELGKEFFP RVAYARNELA ENIIYLDFRS GYRFCKDFPG MLDYRQRAIK RHYIEMTIREKKAKEFLNYI DEEGRITTRG KAVEVVRDVV PHVAIAKRLA TEYDPRKHKY QSSQQTGLDADGKPIIRIFK IKAIKGERHG QDVPAMRGKI GDEELKLLAF ITWKNIDLPY GDNFDLPYLIAVEIKGRIHF KLGAEEIAAI MEAELAKLIG PNKPDEEEYK LYPSIIVTHN FIPSILGDLILLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSE EKIAKYRVPL ARGIKVKPGTDPDYYIENQV WLKR; KENGEFKIEL KTVRVLDAVK REHPAVVDIY DIETFYHEGD VDVVSNEREMKRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSS RRLRTTYLGG VSPDTLEKEGAMRQDIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGF IAKETQAKVL EKLVIHEQITIISYIVLKGS LPAVLRILEA DPHFQX¹YIYA VRKKFLGREV EYDIPFAKRY EFGKGEIIMIIKRFVQVVKE VLGRDKEHPE NLPTYTLEAV AQYSMEDARA TGNLVEWYLL YVKEPEKGLWCKNYDVAPIV KSTIDPIEKK ECAESVTAWG IPGEKPELIK FVTKKRYAVI EAILKEGSVERDLKDYKAIG GKISDRVILL FGYRKEDLRY wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H. 151 P36X¹ K764X² variantof Thermococcus litoralis DNA polymerase, sequence 2 (acc. ADK47977.1)MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARV KDPDVIITYNPKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYERNELA ENIIYLDFRS SYRFCKDFPGMLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRG KAVEIVRDVL PHVAIAKRLATEYDPEKHKY QSSXQTGLDA DGKPIIRIFK IKAIKGERHG QDVPAMRDKI GDEELKLLAFITWKNIDLPY GDNFDLPYLI AVEIKGRIHF KLGAEEIAAI MEAELAKLIG PNKPDEEEYKLYPSIIVTHN FIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLEL LEVVRRDWSEEKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR; KENGEFKIEL KSVRVVDAVK KEHPAVIDIYDIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTI WETEESMKKL QSVWDVSRSSRRLRTTYLGG VSPDTLEKEG AMRQEIKKKM GYPKARWYSK YADTDGFYAT EYEGFYLRGFIAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA DPHFQX¹YIYA VKKKFLGREVEYDIPFAKRY EFGKGEIIMI IKRFVQVVKE VLGRDKENPE NLPTYTLEAV AQYSMEDARATGNLVEWYLL YVKEPEKGLW CENYDIAPIV KATIDPVERK ECAESVTAWG ISGEKPEIIKFVTKKRYAVI EAILKDGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRY wherein X¹ is anyamino acid other than P; in some embodiments, X¹ is selected from Q, N,H, S, T, Y, C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H; andwherein X² is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P,and G; in some embodiments, X² is selected from Q and N. 152 P36X¹ K764Qvariant of Thermococcus litoralis DNA polymerase, sequence 2 (acc.ADK47977.1) MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME SYADEEEARVKDPDVIITYN PKIQRMGDSF YEAVLGKTKS TYELGKEFFP RVAYERNELA ENIIYLDFRSSYRFCKDFPG MLDYRQRAVK RHYIEMTIKE KKAREFLNYI DEEGRITTRG KAVEIVRDVLPHVAIAKRLA TEYDPEKHKY QSSQQTGLDA DGKPIIRIFK IKAIKGERHG QDVPAMRDKIGDEELKLLAF ITWKNIDLPY GDNFDLPYLI AVEIKGRIHF KLGAEEIAAI MEAELAKLIGPNKPDEEEYK LYPSIIVTHN FIPSILGDLI LLANSYYGYM IEEKFGFKVL NSKLPGLLELLEVVRRDWSE EKIAKYRVPL ARGIKVKPGT DPDYYIENQV WLKR; KENGEFKIEL KSVRVVDAVKKEHPAVIDIY DIETFYHEGD VDVVSNEREM KRAEKLGVRL DLFPVVRRTI WETEESMKKLQSVWDVSRSS RRLRTTYLGG VSPDTLEKEG AMRQEIKKKM GYPKARWYSK YADTDGFYATEYEGFYLRGF IAKETQAKVL EKLVIHEQIT IISYIVLKGS LPAVLRILEA DPHFQX¹YIYAVKKKFLGREV EYDIPFAKRY EFGKGEIIMI IKRFVQVVKE VLGRDKENPE NLPTYTLEAVAQYSMEDARA TGNLVEWYLL YVKEPEKGLW CENYDIAPIV KATIDPVERK ECAESVTAWGISGEKPEIIK FVTKKRYAVI EAILKDGSVE RDLKDYKAIG GKISDRVILL FGYRKEDLRYwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H. 153 P36X¹ R761X² variant of Thermococcusgorgonarius DNA polymerase MILDTDYITE LLKDDSAIED EVWKLYFTHP LIDKGLIPMESYADEEGARV KDPDVLITYN IQRMGDRFAV AIFGQPKEKV ELGKEFFPME AYERNELAPNDGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAF ITWKNIDLPY GDNFDFAYLKEVKGRIHFDL YAEEIAQAWE AQLSRLVGQS KPDERELARR KENGEFKIDY TTVRVVRAEKKEHPAVVDIY DIETLYHEGE VDVVSTEKEM KRSEKLGVKF YPVIRRTINL TGEGLERVARLWDVSRSSTG RESYAGGYVK DRNFEX¹YIYA VKKKFLGRPI EYDIPFAKRY EFAEGPILMIIKRFLKVVKE ILGREGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENIVYLDFRSLYP FCKDFPGFIP YRQRAIKILA IETTIREIEE KEFLDYINAK DKITTRGLEIRIVKEVTEKL AVAKRLAARG DPAKHKYDAE XQVGLGAWLK SIIITHNVSP SLLGDLLEERNSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAK SKYEVPPEKL IKIRPGTVISYYIENQVLPA PKT; DTLNREGCEE QKVKKKMKAT KARWYCKECA TDGFFATIPG GFYKRGFFVTETQARVLEAI VIYEQITRDL YIVLKGSGRI VERILRAFGY YDVAPQVGHK IDPIEKKLLDESVTAWGRQY ADAETVKKKA KKKYAVIDEE LKHGDVEEAV KDYKATGPHV GDRAIPFDEFRKEDLRYQKT wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H; and wherein X² is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; in some embodiments, X²is selected from Q and N. 154 P36X¹ R761Q variant of Thermococcusgorgonarius DNA polymerase MILDTDYITE LLKDDSAIED EVWKLYFTHP LIDKGLIPMESYADEEGARV KDPDVLITYN IQRMGDRFAV AIFGQPKEKV ELGKEFFPME AYERNELAPNVYLDFRSLYP FCKDFPGFIP YRQRAIKILA IETTIREIEE KEFLDYINAK DKITTRGLEIRIVKEVTEKL AVAKRLAARG DPAKHKYDAE QQVGLGAWLK DGKPVIRIFK VKKITAERHGQDVPAIRDKI GDEELKMLAF ITWKNIDLPY GDNFDFAYLK EVKGRIHFDL YAEEIAQAWEAQLSRLVGQS KPDERELARR SIIITHNVSP SLLGDLLEER NSFYGYYGYA KFGFKVLYADLPGLLELEYE VRRDWSEIAK SKYEVPPEKL IKIRPGTVIS YYIENQVLPA PKT; KENGEFKIDYTTVRVVRAEK KEHPAVVDIY DIETLYHEGE VDVVSTEKEM KRSEKLGVKF YPVIRRTINLTGEGLERVAR LWDVSRSSTG RESYAGGYVK DTLNREGCEE QKVKKKMKAT KARWYCKECATDGFFATIPG GFYKRGFFVT ETQARVLEAI VIYEQITRDL YIVLKGSGRI VERILRAFGYDRNFEX¹YIYA VKKKFLGRPI EYDIPFAKRY EFAEGPILMI IKRFLKVVKE ILGREGSEPKPTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENI YDVAPQVGHK IDPIEKKLLDESVTAWGRQY ADAETVKKKA KKKYAVIDEE LKHGDVEEAV KDYKATGPHV GDRAIPFDEFRKEDLRYQKT wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 155 P36X¹ R761X² variant ofThermococcus kodakarensis DNA polymerase MILDTDYITE LLKDDSAIEEEVWKLYFTHP LIDKGLVPME SYADEEGARV KDPDVLITYN IQRMGDRFAV AVFGQPKEKVELGKEFLPME AYERNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILA ITMTIKEIEEMEFLKYINAK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHKYDAE XQVGLSAWLKDGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAF ITWKNVDLPY GDNFDFAYLKEVKGRIHFDL YAEEITTAWE AQLSRLIGQS KPDEKELARR SIIITHNVSP SLLGDLLEERNSYYGYYGYA KYGFKVIYSD LPGALELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVISYYIENQVLPA PKGT; KENGEFKIEY TVVTVKRVEK REHPAVIDIY DIETLYHEGE VDVVSTEREMKRCEKLGINF YPVIRRTINL TGENLERVAR LWDVSRSSTG RQSYEGGYVK DTLNREGCKEQKIKKKMKAT RARWYCKECA TDGFFATIPG GFYKRGFFVT ETQARVLEAL VIHEQITRDLYIVLKGSGRI VERILRAFGY DRTFEX¹YFYA VQKKFLGRPV EYDIPFAKRY EFAEGPILMIIKRFLRVVKE ALGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENIYDVAPQVGHR IDPIERKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKDGDVEKAVKDYKATGPHV GDRAIPFDEF RKEDLRYQKT wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H; and wherein X² isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 156 P36X¹ R761Q variantof Thermococcus kodakarensis DNA polymerase MILDTDYITE LLKDDSAIEEEVWKLYFTHP LIDKGLVPME SYADEEGARV KDPDVLITYN IQRMGDRFAV AVFGQPKEKVELGKEFLPME AYERNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILA ITMTIKEIEEMEFLKYINAK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHKYDAE QQVGLSAWLKDGKPVIRIFK VKKITAERHG QDVPAIRDKI GDEELKMLAF ITWKNVDLPY GDNFDFAYLKEVKGRIHFDL YAEEITTAWE AQLSRLIGQS KPDEKELARR SIIITHNVSP SLLGDLLEERNSYYGYYGYA KYGFKVIYSD LPGALELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVISYYIENQVLPA PKGT; KENGEFKIEY TVVTVKRVEK REHPAVIDIY DIETLYHEGE VDVVSTEREMKRCEKLGINF YPVIRRTINL TGENLERVAR LWDVSRSSTG RQSYEGGYVK DTLNREGCKEQKIKKKMKAT RARWYCKECA TDGFFATIPG GFYKRGFFVT ETQARVLEAL VIHEQITRDLYIVLKGSGRI VERILRAFGY DRTFEX¹YFYA VQKKFLGRPV EYDIPFAKRY EFAEGPILMIIKRFLRVVKE ALGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWENIYDVAPQVGHR IDPIERKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKDGDVEKAVKDYKATGPHV GDRAIPFDEF RKEDLRYQKT wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H. 157 P36X¹ K761X² variantof Thermococcus species 9°N-7 DNA polymerase MILDTDYITE LLKDDSAIEDEVWKLYFNHP LIDKGLIPME SYADGSEARV KDPDVLITYN IQRMGDRFAV AVFGKPKEKVELGREFFPME AYKRNELAPN VYLDFRSLYP FCKDFPGFIP YRQRAIKILA IEMVIRELEEKEFLKYINPK GKITTRGLEI RIVKEVTEKL AVAKRLAARG DPTKHRYDAE XQVGLGAWLKNGKPVIRVFK VKKVTAKRHG QDVPAIRDRI GDEELTMLAF ITWKKIDLPY GDNFDFAYLKEVKGRIHFDL YAEEIAQAWE AQLSRLIGQS KPDERELARR SIIITHNVSP SLLGDLLEERNSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAK SKYEVPPEKL VKIRPGTVISYYIENQVLPA VKGKK; KENGEFKIEY TVVKVKRAEK RAHPAVVDIY DIETLYHEGE VDVVSTEKEMKRCEELGIKF YPVIRRTINL SGEGLERVAR LWDVSRSSTG RGGYAGGYVK DTLNREGCKEQKIKRKMKAT KARWYCKECA TDGLHATIPG GFYVRGFFVT ETQARVLEAI VIHEQITRDLYIVLKGSGRI VERILKAFGY DRTFEX¹YFYA VQKKFLGRPI EYDIPFAKRY EFGTGPILMIIKRFLRVVRE TLGRDGSEPK PTYTLEAVYE YSMEDAKVTY NLVEWFLLRK EPERGLWDNIYDVAPEVGHK VDPLEKKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKHGDVEEAVRDYKATGPHV GDRAIPADEF RKEDLRYQKT wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H; and wherein X² isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, P, and G; insome embodiments, X² is selected from Q and N. 158 P36X¹ K761Q variantof Thermococcus species 9°N-7 DNA polymerase MILDTDYITE LLKDDSAIEDEVWKLYFNHP LIDKGLIPME SYADGSEARV KDPDVLITYN IQRMGDRFAV NGKPVIRVFKVKKVTAKRHG QDVPAIRDRI GDEELTMLAF ITWKKIDLPY GDNFDFAYLK EVKGRIHFDLKENGEFKIEY TVVKVKRAEK RAHPAVVDIY DIETLYHEGE VDVVSTEKEM KRCEELGIKFYPVIRRTINL DRTFEX¹YFYA VQKKFLGRPI EYDIPFAKRY EFGTGPILMI IKRFLRVVRETLGRDGSEPK PTYTLEAVYE AVFGKPKEKV ELGREFFPME AYKRNELAPN VYLDFRSLYPFCKDFPGFIP YRQRAIKILA IEMVIRELEE KEFLKYINPK GKITTRGLEI RIVKEVTEKLAVAKRLAARG DPTKHRYDAE QQVGLGAWLK YAEEIAQAWE AQLSRLIGQS KPDERELARRSIIITHNVSP SLLGDLLEER NSFYGYYGYA KFGFKVLYAD LPGLLELEYE VRRDWSEIAKSKYEVPPEKL VKIRPGTVIS YYIENQVLPA VKGKK; SGEGLERVAR LWDVSRSSTG RGGYAGGYVKDTLNREGCKE QKIKRKMKAT KARWYCKECA TDGLHATIPG GFYVRGFFVT ETQARVLEAIVIHEQITRDL YIVLKGSGRI VERILKAFGY YSMEDAKVTY NLVEWFLLRK EPERGLWDNIYDVAPEVGHK VDPLEKKLLD ESVTAWGREY ADAETVKKKA KKKYAVIDEE LKHGDVEEAVRDYKATGPHV GDRAIPADEF RKEDLRYQKT wherein X¹ is any amino acid other thanP; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W,A, I, L, F, V, G; in some embodiments, X¹ is H. 159 P40X¹ E775Q variantof Pyrobaculum calidifontis DNA polymerase MRFWPLDATY YFYAKCDKCDFLKVVAKVPE IDKGVVPCAW PPLRVLAFDI FEAEGRDDRR SERAKALGVP IVDEFPEIKVNDPAKRPTLM PLDQVAAASV KGAIVLEPKP EPHEPDPPEG RAVREEAKKY VGARWYKKEVTDSLFVKKSG AKKRYAGLLR ILKSKSVGEA LDKELDEYKA GPGKVSERAM VLGVKESDLKSVVGGVPEVR ASLAKSYLSR DVRKLREAAL NVVEAREAGK EVYNERGSPD VIRGFVDFVKLRVDRLGGVP KTLDRVAEYF RYVLDDVRST GNRVEWMLLR GLYSDVLVLD VVVAPEVGHRPPDSPEYRLL AESVTAFARA AVDRLVKYVE DGRIDIVGFE RERVVKYVRE YGPHVHAALEPYIFVDDASK TGRVQKSLLD VFGVDGEGRR VAPVEAVEVV GAPGVVDVYE LGPLPLYEVVPLRDPVVMLA EFDPDVIVGY QQSVYGHWSV GVMKRSERVL LGLAEKLLPF YAYRMGEVAPFSSMYPNIMM FRKAPTGFIP DERQRALKVM ILLDVVEYAK ERHGIEIKVD VVRGDWCELAVVERLKAYKF LKRRGYKVGK VDVDYYIEKQ FLG; VVLVDRRFRX¹ ERRFFGRPTI ADIRYYMRYMEWAGVEEGFP VKTSDGREEV NSNGFDWPYL VGRANVDLYN IPGHKVYEYW LIQLSSVSGLNREEREYEPY KYNLSPDTYL AVLKHLVELR ANAMYGYLGW RLGIEVIYGD KDYERVLFTEKEVQLNVVEL DLDDLIIWKT GTTVGYVIVR VIPAALRIAE wherein X¹ is any amino acidother than P; in some embodiments, X¹ is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H. 160 P42X¹ E778Qvariant of Pyrobaculum aerophilum DNA polymerase MKFKLWPLDA RX¹YFYADCPARSFLKIVARV YMLDMGVVPC GFPPPLRVLA VEVFEASGRD PYLAERARAL LYNIVDEFPEEYWRDQGKRP SGLPLDQVAA EPYKGAIVLE TYLERGEPDP ELRKRVREEL TGWVGARWYKYGDTDSLFVK FTEAKKRYAG IELILTSRDV WKTLDKELDE VVKGGEKVSE IAEVIGIKEGTYSVVGGVPE CDPESVRSQL PEDVRKLREA SWNTVDAEAT FDIEVYNERG DRSVLRSFIDGIPLKVDRVG IKLKTLDRVA LLRQYVIDDV ASVGNRVEWM PRPGLYSDVL PGGVYVAPEVKKYPPDSPEY KEVAESVTAF KSGDVEKLVK LLRDGRIDIV SEARQKVVKY YKAYPPHVHARAVPYIFIDD DLKTGRSQRT VRIFGISESG GRVAPVEEVV AAALPGVSGV GEKLGNLPVYTPDPLRDPVI FVREFDPDVI GAPQQSVYGH EYFGVMKREE KSTYGLAEKL LLRYAYRLGEALDFSSMYPN GHRFRREPPG RVLDERQRAL ARAILKDVIE YVEEKYGIDI GFEVVRGDWSVRGVIDKLRN AILLKKRGYK IEKIDLDYYV LLDFF; DRVVVVDRRF AVERRYLGRP YEADIRFYMRKVAEWGGVTE LLAVQASDGR VGYNSNQFDW WSVTGRANVD RVLVPGHKIY LPFLIQLSSVVAPNREEREY IMMKYNLSPD FIPLVLRQLI KIMANAMYGY YARKAGIVVI KIDKDYSTVLELAKEVQLRV YEVDLDDLII VGKGTTIGYV ERQVIPAALR wherein X¹ is any amino acidother than P; in some embodiments, X¹ is selected from Q, N, H, S, T, Y,C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H. 161 PyrococcusDNA polymerase sequence including exonuclease domain and catalyticdomain, P36X¹ MILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKVKDPDIIITYN MQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALYKFCKDFPGFI DYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQKVAVAKRLAAK YDPRKHKYDA TKQTGL; EGKPVIRLFK VKKITAERHG QDVPTIREKIGDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP KENGEFKIEH KIVRIVDAEKREHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKL YHVIRRTINL TGEGLERVAKLWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKA AKARWYCKEC DTDGLYATIPEGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGP AVLRILEGFG DRTFRX¹YIYAVEKKFLGRPI EYDIPFAKRY EFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYEYSMEDAKATY NLVEWFLLRK KEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGREGGKSEEIKKK TKKKYALIDE ILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQKwherein X¹ is any amino acid other than P; in some embodiments, X¹ isselected from Q, N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in someembodiments, X¹ is H. 162 Pyrococcus DNA polymerase N-terminal domaincomprising a uracil-binding pocket, P36X¹ MILDADYITE LLKDDSKIEETVWRLYFEHP LIDKGLIPME EGKPVIRLFK VKKITAERHG QDVPTIREKI G; KENGEFKIEHKIVRIVDAEK REHSAVVDIF DRTFRX¹YIYA VEKKFLGRPI EYDIPFAKRY wherein X¹ isany amino acid other than P; in some embodiments, X¹ is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H.163 Pfu DNA polymerase (GenBank Acc. No. WP_011011325.1) N-terminaldomain comprising a uracil-binding pocket, P36X¹ MILDVDYITE LLRDDSKIEETVWKLYLEHP LIDKGLIPME EGKPVIRLFK VKKITGERHG QDVPTIREKV G; KENGKFKIEHKIVRIVDVEK REHPAVVDIF DRTFRX¹YIYA VEKKFLGKPI EYDIPFAKRY wherein X¹ isany amino acid other than P; in some embodiments, X¹ is selected from Q,N, H, S, T, Y, C, M, W, A, I, L, F, V, G; in some embodiments, X¹ is H.164 Deep Vent DNA polymerase N-terminal domain comprising auracil-binding pocket, P36X¹ MILDADYITE LLKDDSQIDE EVWRLYFEHP LIDKGLIPMEDGKPIIRIFK VRKITAERHG QDVPAIRDKI G; KENGEFKVEY KIVRIIDAEK REHSAVIDIFDRNFRX¹YIYA VRKKFLGRPI EYDIPFAKRY wherein X¹ is any amino acid otherthan P; in some embodiments, X¹ is selected from Q, N, H, S, T, Y, C, M,W, A, I, L, F, V, G; in some embodiments, X¹ is H. 165 Thermococcuslitoralis DNA polymerase N-terminal domain comprising a uracil-bindingpocket, P36X¹ MILDTDYITK LLKDDSAIEE EVWKLIFEHP LIDKGLIPME DGKPIIRIFKIKAIKGERHG QDVPAMRGKI G; KENGEFKIEL KTVRVLDAVK REHPAVVDIY DPHFQX¹YIYAVRKKFLGREV EYDIPFAKRY wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 166 Thermococcus gorgonarius DNApolymerase N-terminal domain comprising a uracil-binding pocket, P36X¹MILDTDYITE LLKDDSAIED EVWKLYFTHP LIDKGLIPME DGKPVIRIFK VKKITAERHGQDVPAIRDKI G; KENGEFKIDY TTVRVVRAEK KEHPAVVDIY DRNFEX¹YIYA VKKKFLGRPIEYDIPFAKRY wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 167 Thermococcus kodakarensis DNApolymerase N-terminal domain comprising a uracil-binding pocket, P36X¹MILDTDYITE LLKDDSAIEE EVWKLYFTHP LIDKGLVPME DGKPVIRIFK VKKITAERHGQDVPAIRDKI G; KENGEFKIEY TVVTVKRVEK REHPAVIDIY DRTFEX¹YFYA VQKKFLGRPVEYDIPFAKRY wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 168 Thermococcus species 9°N-7 DNApolymerase N-terminal domain comprising a uracil-binding pocket, P36X¹MILDTDYITE LLKDDSAIED EVWKLYFNHP LIDKGLIPME NGKPVIRVFK VKKVTAKRHGQDVPAIRDRI G; KENGEFKIEY TVVKVKRAEK RAHPAVVDIY DRTFEX¹YFYA VQKKFLGRPIEYDIPFAKRY wherein X¹ is any amino acid other than P; in someembodiments, X¹ is selected from Q, N, H, S, T, Y, C, M, W, A, I, L, F,V, G; in some embodiments, X¹ is H. 169 Pyrococcus DNA polymerasesequence including exonuclease domain and DNA binding domain, P36HMILDADYITE LLKDDSKIEE TVWRLYFEHP LIDKGLIPME SYADEEEAKV KDPDIIITYNMQRIGDMTAV AIFGKPKEKV ELGKEFFPME AYERNELAPN IVSLDFRALY KFCKDFPGFIDYRQRAIKIL YIEFVWKELE ALEFVDYINA EGKIITRGLE VRIVKEVTQK VAVAKRLAAKYDPRKHKYDA TKQTGLTSWL KKVWRVGKMI KQKK EGKPVIRLFK VKKITAERHG QDVPTIREKIGDEELKLLAF ITWKKIDLPY GDSFDLPYLA EVKGRIHFDL YADEIAKAWE AQLSRLVGQPKPDEREYERR PSIIITHNVS PSLLKRLLDE ANSYYGYYGY EKFGFKVLYI KLPGLLELEYIVRRDWSEIA LSKYEIPPEK GVKIKPGMVI EYYIENQVLP NIKKSGTGGG SFTYDEGGGKKENGEFKIEH KIVRIVDAEK REHSAVVDIF DIETLYHEGE VEVVSSEREM KRAEKLGIKLYHVIRRTINL TGEGLERVAK LWDVSRSSTG LRESYAGGFV PDTLNREGCR RQKIKTKMKAAKARWYCKEC DTDGLYATIP EGFYKRGFFV KETQARVLEA LAIYEQITRP GYIVLRGDGPAVLRILEGFG GATVKFKYKG TGRGAVSEKD DRTFRHYIYA VEKKFLGRPI EYDIPFAKRYEFGKGPIIMI IKRFLKIIRE TIGRDGSEPK PTYTLEAVYE YSMEDAKATY NLVEWFLLRKKEPEKGLWEN NYDVAPEVGH SQDPIEKIML AESVTAWGRE GGKSEEIKKK TKKKYALIDEILKHGNVEEA LHEYKAIGPH ISNRAILAEE YRKEDLRWQK EEKEVDISKI APKELLQMLE

What is claimed is: 1-55. (canceled)
 56. A nucleic acid comprising asequence encoding a thermophilic DNA polymerase comprising a family Bpolymerase N-terminal domain comprising a uracil-binding pocket and afamily B polymerase catalytic domain, the family B polymerase N-terminaldomain comprising a uracil-binding pocket having an amino acid sequencein which the position corresponding to position 36 of SEQ ID NO: 1 isany amino acid other than P, and the family B polymerase catalyticdomain having an amino acid sequence in which the position correspondingto position 762 of SEQ ID NO: 1 is a neutral amino acid residue, whereinthe thermophilic DNA polymerase comprises an amino acid sequence havingat least 90%, 95%, 98%, 99%, or 100% identity to a sequence selectedfrom SEQ ID NOs: 80 to 113 and 127 to 160, wherein X¹ is any amino acidother than P and X² is the neutral amino acid residue.
 57. An expressionvector comprising the nucleic acid of claim
 56. 58. An isolated hostcell comprising the expression vector of claim
 57. 59. A method ofproducing a thermophilic DNA polymerase, comprising culturing at leastone host cell comprising a the nucleic acid of claim 56, wherein the atleast one host cell expresses the thermophilic DNA polymerase.
 60. Themethod of claim 59, further comprising isolating the thermophilic DNApolymerase.
 61. A composition comprising the nucleic acid of claim 56.62. The composition of claim 61, comprising at least one hot startinhibitor.
 63. The composition of claim 61, comprising at least two hotstart inhibitors.
 64. The composition of claim 62, wherein each hotstart inhibitor is independently selected from an antibody, anAffibody®, a chemical modification, and an oligonucleotide.
 65. Thecomposition of claim 63, wherein the composition comprises at least twoantibodies.
 66. The composition of claim 63, wherein the compositioncomprises an antibody and an oligonucleotide. 67-104. (canceled)
 105. Anisolated host cell comprising the nucleic acid of claim
 56. 106. Thecomposition of claim 63, wherein each hot start inhibitor isindependently selected from an antibody, an Affibody®, a chemicalmodification, and an oligonucleotide.
 107. The composition of claim 64,wherein the composition comprises at least two antibodies.
 108. Thecomposition of claim 64, wherein the composition comprises an antibodyand an oligonucleotide.